Review ArticleFrom Structure-Function Analyses to Protein Engineering forPractical Applications of DNA Ligase

Maiko Tanabe1 Yoshizumi Ishino2 and Hirokazu Nishida1

1Central Research Laboratory Hitachi Ltd 1-280 Higashi-koigakubo Kokubunji Tokyo 185-8601 Japan2Department of Bioscience and Biotechnology Graduate School of Bioresource and Bioenvironmental SciencesFaculty of Agriculture Kyushu University 6-10-1 Hakozaki Higashi-ku Fukuoka-shi Fukuoka 812-8581 Japan

Correspondence should be addressed to Yoshizumi Ishino ishinoagrkyushu-uacjpand Hirokazu Nishida hirokazunishidaabhitachicom

Received 20 February 2015 Accepted 18 May 2015

Academic Editor Frederic Pecorari

Copyright copy 2015 Maiko Tanabe et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

DNA ligases are indispensable in all living cells and ubiquitous in all organs DNA ligases are broadly utilized in molecular biologyresearch fields such as genetic engineering and DNA sequencing technologies Here we review the utilization of DNA ligases ina variety of in vitro gene manipulations developed over the past several decades During this period fewer protein engineeringattempts for DNA ligases have been made as compared to those for DNA polymerases We summarize the recent progress in theelucidation of theDNA ligationmechanisms obtained from the tertiary structures solved thus far in each step of the ligation reactionscheme We also present some examples of engineered DNA ligases developed from the viewpoint of their three-dimensionalstructures

1 Introduction

DNA ligases are critical DNA replication and repair enzymesthey have beenwidely used inmolecular biology and biotech-nology applications such as cloning and next-generationDNA sequencing [1 2] DNA ligases catalyze the joining ofadjacent 31015840-hydroxyl and 51015840-phosphorylated DNA terminiin duplex DNA All DNA ligases accept nicked dsDNAand homologous cohesive ends as substrates although theminimum length of the overlap required for efficient ligationvaries widely Some ligases most notably T4 DNA ligase alsoaccept fully base-paired (blunt end) substrates for in vitroligation reactions

DNA ligases share a common mechanism and a highdegree of structural similarity with other members of thenucleotidyltransferase superfamily including RNA ligasesand RNA-capping enzymes [3] Like other nucleotidyltrans-ferases DNA ligases utilize an ATP molecule to activate theenzyme and thus DNA ligases are prepared to react withDNA substrates [4]

The details of each step in the DNA ligation reactionsequence have been clarified by many efforts performed by

numerous researchers Above all Shumanrsquos group has madeextensive contributions toward the elucidation of the DNAligation mechanisms [5] We describe the exemplified DNAligation schemes in particular in the second section of thisreview

Since the discovery of DNA ligase this enzyme has beenwidely utilized in several types of molecular biology andbiotechnology applications DNA ligase is frequently utilizedin combination with restriction enzymes in recombinantDNA experiments The enzyme has been applied in DNAsequencing methods in diverse ways by taking advantage ofthe fact that DNA ligase does not require dNTPs as substratesfor its function since large amounts of dNTPs in solution ageneral situation in DNA polymerase reactions sometimesinhibit effective measurements and cause misinterpretationsDNA ligase is also utilized in analytical methods for protein-protein interactions as developed by Landegren et al [6]These methods are widely applied for quick and effective insitu investigations of protein-protein interactions [7] We willdescribe the details of the utilization of DNA ligase in severalaspects of genetic engineering technology andmolecular andcell biology in the third section

Hindawi Publishing CorporationArchaeaVolume 2015 Article ID 267570 20 pageshttpdxdoiorg1011552015267570

2 Archaea

Since the first report of the X-ray crystal structure ofthe ATP-dependent DNA ligase from bacteriophage T7 [8]the crystallographic analyses of the archaeal and eukaryoticATP-dependent DNA ligases were subsequently reportedduring the first decade of the new century [9ndash11] The ATP-dependent DNA ligases fromArchaea and Eukarya comprisethree domains but surprisingly the relative arrangementsof the three domains were completely different from eachother in these reports [9ndash11] reflecting the dynamic domainmotion in the 3-step ligation sequence Based on these struc-tural transitions in the ligation sequence several attemptstoward improving the enzymatic reaction have been made bymutating the enzymes In the fourth and the fifth sections wesummarize the structural information about DNA ligase andthe mutation-based improvements of its enzymatic efficacy

2 Basis of the DNA Ligase Reaction

The Gellert Lehman Richardson and Hurwitz laboratoriesdiscovered DNA ligases in 1967 and 1968 [12ndash15] By joiningthe 31015840-OH and 51015840-phosphate termini (nicks) to form aphosphodiester bond DNA ligases play an important rolein maintaining genome integrity They are essential for DNAreplication and repair in all organisms [16 17] FurthermoreDNA ligases have long been a critical reagent in the develop-ment of molecular cloning and many subsequent aspects ofDNA biotechnology

All DNA ligation reactions entail sequential nucleotidyl-transfer steps [18] The reaction mechanism can be dividedinto three distinct catalytic events the first (step 1) the acti-vation of the enzyme through the covalent addition of AMPto the conserved catalytic lysine of the ligase accompanied bythe release of PPi or nicotinamide mononucleotide from thecofactor (ATP or NAD+) the second (step 2) the binding ofthe ligase-adenylylate to the substrate DNA and the transferof AMP from the ligase to the 51015840-phosphoryl group of thenick on the DNA and the third (step 3) the formation ofthe phosphodiester-bond with the concomitant release offree AMP from the DNA-adenylylate intermediate (Figure 1)All three chemical steps depend on a divalent cation TheDNA ligases are grouped into two families according to thecofactor dependence for the reaction ATP-dependent DNAligases are found in viruses bacteria Eukarya and Archaea[18 19] whereas NAD+-dependent DNA ligases are primarilypresent in bacteria and entomopox viruses [19 20]

Sequence analyses ofDNA ligases revealed that they sharesix motifs (I III IIIa IV V and VI) which are also conservedin the nucleotidyltransferase superfamily members includ-ing RNA ligase [21] tRNA ligase [22] and mRNA-cappingenzymes [23] except for motif VI In the ATP-dependentDNA ligases these six motifs align well among the enzymesfrom viruses bacteriophages Archaea and Eukarya withoutlong gaps or insertions (Figure 2)The sequences of the ATP-dependent DNA ligases showed that the amino acids inmotifs I III IIIa IV V and VI contact the substrates AMPor DNA The important roles of the individual amino acidsin these motifs were verified by alanine scanning mutationalstudies and confirmed as essential for one or more steps ofthe ligation pathway [24] For example motif I (KxDGxR)

Ligase ATP

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Figure 1 Schematic diagram of the three-step reaction catalyzed byATP-dependent DNA ligases The three-step reaction catalyzed byDNA ligase (Ligase) results in the serial transfer of AMP (adenosine51015840-monophosphate) to an active site lysine (step 1) and then to the 51015840-PO4end of DNA (step 2) During step 3 the 31015840-OH end of a second

DNA strand attacks the 51015840-PO4 to release AMP and generate the

ligated DNA product

contains the lysine that becomes covalently linked to AMPin the first step of the ligase reaction [25 26] Furthermorethe arginine and lysine side chains in motif VI (RxDK) orientthe PPi leaving group apically relative to the attacking motifI lysine during step 1 enzyme nucleotidylation reaction [2728]

3 Utilization of DNA Ligases in GeneticResearch Technologies

The in vitro manipulation of DNA has been broadly appliedto studies in molecular genetics microbiology immunol-ogy and oncology and also to practical uses in clinicalassays [29]This technologywas developedwithDNA-relatedenzymes which were discovered by research on the molecu-lar mechanisms of the replication and repair of DNA DNAreplication is initiated by DNA primase which synthesizessmall RNA primers that are subsequently elongated by DNApolymerases [30] Based on its primer extension activity aDNA polymerase can be utilized in the polymerase chainreaction (PCR) which amplifies a single copy of DNA byseveral orders of magnitude in a brief in vitro reaction [31]In addition DNA ligase plays a role in joining Okazakifragments during lagging strandmaturation [32] Based on itsactivity to join twoDNA strands in vitro DNA ligase has beencontributing to recombinant DNA technology for exampleDNA cloning [33] In addition DNA ligase is broadly utilizedfor ligase-mediated mutation detection methods and DNAsequencing

31 Oligonucleotide Ligation Assay (OLA) DNA ligase canonly join two DNA strands when they are perfectly

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middot middot middot------AITKPLLAATLENIEDVQF------PCLATPKIDGIRSVKQ--T-middot middot middot

middot middot middot----VNIKTNPFKAVSFV-ESAIKKALDNAGYLIAEIKYDGVRGNICVDNTmiddot middot middot

middot middot middotKVQVQLGKPIKPMLAQQAASIRDALLEMGG-EAEFEIKYDGARVQVHKD-Gmiddot middot middot

middot middot middotTLKPQVGIPIRPMLAERLSNPEEILKKMGG-NAIVDYKYDGERAQIHKK-Emiddot middot middot

middot middot middotHCKLSPGIPLKPMLAHPTRGISEVLKRFEEAAFTCEYKYDGQRAQIHALEGmiddot middot middot

middot middot middotEGSDGEISIEGATFQD----

middot middot middotFMLDGELMVKGVDFNTGSGLmiddot middot middot

middot middot middot

middot middot middotAIVEGELVAIG-ENGRPLPFmiddot middot middot

middot middot middotFIIEGEIVAIDPESGEMRPFmiddot middot middot

middot middot middotFILDTEAVAWDREKKQIQPFmiddot middot middot

middot middot middotYWFDYVT-----Dmiddot middot middot

middot middot middotKLYAILPLHIVESmiddot middot middot

middot middot middotNLFDVLYVDGQSLmiddot middot middot

middot middot middotFLFDLMYYEDVDYmiddot middot middot

middot middot middotYAFDLIYLNGESLmiddot middot middot

middot middot middotLLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMK----QFKDAEATmiddot middot middot

middot middot middotLQQLYEQKRAEGHEGLIVKDP--MCIYKR-GKKS-GWWKMK------PENEADmiddot middot middot

middot middot middotAEAFYKRALEMGHEGLMAKRL--DAVYEP-GNRGKKWLKIK-----PTMENLDmiddot middot middot

middot middot middotLKSFFYRAISEGGEGVMVKAIGKDAIYQA-GARGWLWIKLKRDYQSEMADTVDmiddot middot middot

middot middot middotIAEFLEQSVKDSCEGLMVKTLDVDATYEI-AKRSHNWLKLKKDYLDGVGDTLDmiddot middot middot

middot middot middotPRFPVFIGIRHEEDR-----------------------------------

middot middot middotLRHPSFVMFRGTEDNPQEKM------------------------------

middot middot middotPRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES----------

middot middot middotPRFIR-W--RDDKSPEDATTTDEILEMYNKQPKKKIESPAVDESV-----

middot middot middotPRFIR- REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

ChV 22

T7 29Pfu 199

Sso 229

Hu 281

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T7 87Pfu 272

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T7 46Pfu 336

Sso 367

Hu 246

ChV 134

T7 196Pfu 372

Sso 407

Hu 467

ChV 284

T7 339Pfu 525

Sso 580

Hu 873

Figure 2 Conserved sequence elements define a superfamily of covalent nucleotidyltransferases Six collinear sequence elements designatedas motifs I III IIIa IV V and VI are conserved in capping enzymes and ligases The amino acid sequences are aligned for the DNA ligasesfrom Chlorella virus (ChV) bacteriophage T7 (T7) Pyrococcus furiosus (Pfu) Sulfolobus solfataricus (Sso) and human (Hu) The number ofintervening amino acid residues is indicated Conserved residues are highlighted in red (nucleotidyltransferases) and blue (DNA and RNAligases)

hybridized to a complementary DNA sequence Even asingle base pair mismatch between two strands significantlydecreases the efficiency of the strand joining reaction [34]In 1988 the Oligonucleotide Ligation Assay (OLA) wasdeveloped as a useful method to detect the genotype of thetarget DNA by utilizing this characteristic [6 35] This wasthe first example of the development of a method for geneanalysis by using DNA ligase

OLA consists of two steps First the two probes to behybridized with the target DNA at the sequence of interestmust lie directly adjacent to one another In this case oneprobe must contain a reporter group which is either 32P- orfluorescently labeled and the other must include a recogni-tion group for immobilization such as a biotin group which

could be captured by streptavidin immobilized on a solidsupport Second the neighboring probes that are perfectlyhybridized to the target DNA at the sequence of interestare joined by DNA ligase Then the ligation signal is subse-quently captured by streptavidin and detected by autoradio-graphy or fluorography (Figure 3)This assay takes advantageof the enzymatic accuracy in two events the hybridizationof the probes and the joining by DNA ligase resulting in thereliable analysis of the clinical genotype

A breakthrough in OLA for practical use occurred in1990 when PCRwas coupled to the assay prior to the ligationreaction (PCR-OLA) [36] PCR-OLA amplification only forthe target DNA enhanced the sensitivity of the assay enabl-ing the nonradioactive detection of theOLA results Recently

4 Archaea

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Figure 3 Schematic diagrams for the Oligonucleotide Ligation Assay (OLA) DNA fragments from the normal type and the mutant type(at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) Denature samples are heated to 85ndash95∘C forone to several minutes to denature (separate into single strands) the target DNA (2) Hybridization two oligonucleotide probes 51015840-biotinlabeled probes and 31015840-fluorescent (or radiolabeled) probes which are complementary to the normal-type target hybridize to the target DNAfragment (3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase (4) Binding tosupport the fluorescently labeled ligation samples are immobilized to streptavidin and detected by fluorography only if ligated to biotinylatedoligonucleotides that can be bound to streptavidin on a solid support (left)

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Figure 4 Schematic diagrams for the Ligation Detection Reaction (LDR) processes DNA fragments from the normal type and the mutanttype (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) The denaturation process is the same as inFigure 3 (1) (2) Hybridization two oligonucleotide probes complementary to the normal-type target hybridize to the target DNA fragment(3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase and thermocycling linearlyamplifies the amount of product formed In the case of the mutant type template the presence of a single-base mismatch at the junctioninhibits the ligation and therefore no ligated LDR probes are formed (right)

a sensitive specific and high-throughput OLA was devel-oped for the detection of genotypic human immunodefi-ciency virus type 1 (HIV-1) resistance to a Food and DrugAdministration-approved protease [37] This report revealedthat OLA can be used for the detection of the HIV-1 genotype(wild type or mutant) as a genetic diagnosis method

32 DNA Ligase-Mediated Cycling of the Ligation ReactionFurther spreading of DNA ligase-mediated technologies wasachieved by cycling the ligation reaction like PCR using

a thermostable DNA ligase which became available com-mercially in 1990 The thermostable DNA ligase enables thecycling OLA reaction OLA reaction method with thermalcycling procedure developed by using thermostable DNAligase is often specifically termed LigationDetectionReaction(LDR) (Figure 4)Through repeated denaturation annealingand ligation the target signal is amplified linearly [38ndash40]Ligation Chain Reaction (LCR) [38 39 41] and LigationAmplification Reaction (LAR) [42] are also performed byrepeated cycles of heat denaturation of a DNA template

6 Archaea

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Figure 5 Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes (Here wedescribe LCR) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown asreaction templates (1)The denaturation process is the same as in Figure 3 (1) (2) Hybridization four oligonucleotide probes complementaryto the normal-type target hybridize to the target DNA fragment and (3) Ligation adjacent probes that are perfectly complementary to thetarget (left) are connected by DNA ligase Ligated LCR probes from the first round of the ligation become the targets for the subsequent roundusing another probe set (dotted lines) complementary to the first set Thus the amount of ligated LCR probes increases exponentially In thecase of the mutant type template the presence of a single-base mismatch at the junction inhibits the ligation and therefore no ligated LCRprobes are formed (right)

containing the target sequence annealing of probes andligation processes After annealing the first set of two adjacentoligonucleotide probes to the target DNA sequence in aunique manner a second set of complementary oligonu-cleotide probes that hybridize to the sequence opposite to thetarget DNA sequence is applied (Figure 5)Thereafter a ther-mostableDNA ligase will covalently link each pair of adjacent

probes that are completely hybridized at the junction of theadjacent probes Through the repeated sequential processesincluding denaturation annealing and ligation the targetsignal is amplified exponentially These methods require athermostable DNA ligase to allow the ligation to occurunder temperature conditions that prevent mismatches fromhybridizing to form acceptable substrates The thermostable

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

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Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

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(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

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Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 Archaea

Since the first report of the X-ray crystal structure ofthe ATP-dependent DNA ligase from bacteriophage T7 [8]the crystallographic analyses of the archaeal and eukaryoticATP-dependent DNA ligases were subsequently reportedduring the first decade of the new century [9ndash11] The ATP-dependent DNA ligases fromArchaea and Eukarya comprisethree domains but surprisingly the relative arrangementsof the three domains were completely different from eachother in these reports [9ndash11] reflecting the dynamic domainmotion in the 3-step ligation sequence Based on these struc-tural transitions in the ligation sequence several attemptstoward improving the enzymatic reaction have been made bymutating the enzymes In the fourth and the fifth sections wesummarize the structural information about DNA ligase andthe mutation-based improvements of its enzymatic efficacy

2 Basis of the DNA Ligase Reaction

The Gellert Lehman Richardson and Hurwitz laboratoriesdiscovered DNA ligases in 1967 and 1968 [12ndash15] By joiningthe 31015840-OH and 51015840-phosphate termini (nicks) to form aphosphodiester bond DNA ligases play an important rolein maintaining genome integrity They are essential for DNAreplication and repair in all organisms [16 17] FurthermoreDNA ligases have long been a critical reagent in the develop-ment of molecular cloning and many subsequent aspects ofDNA biotechnology

All DNA ligation reactions entail sequential nucleotidyl-transfer steps [18] The reaction mechanism can be dividedinto three distinct catalytic events the first (step 1) the acti-vation of the enzyme through the covalent addition of AMPto the conserved catalytic lysine of the ligase accompanied bythe release of PPi or nicotinamide mononucleotide from thecofactor (ATP or NAD+) the second (step 2) the binding ofthe ligase-adenylylate to the substrate DNA and the transferof AMP from the ligase to the 51015840-phosphoryl group of thenick on the DNA and the third (step 3) the formation ofthe phosphodiester-bond with the concomitant release offree AMP from the DNA-adenylylate intermediate (Figure 1)All three chemical steps depend on a divalent cation TheDNA ligases are grouped into two families according to thecofactor dependence for the reaction ATP-dependent DNAligases are found in viruses bacteria Eukarya and Archaea[18 19] whereas NAD+-dependent DNA ligases are primarilypresent in bacteria and entomopox viruses [19 20]

Sequence analyses ofDNA ligases revealed that they sharesix motifs (I III IIIa IV V and VI) which are also conservedin the nucleotidyltransferase superfamily members includ-ing RNA ligase [21] tRNA ligase [22] and mRNA-cappingenzymes [23] except for motif VI In the ATP-dependentDNA ligases these six motifs align well among the enzymesfrom viruses bacteriophages Archaea and Eukarya withoutlong gaps or insertions (Figure 2)The sequences of the ATP-dependent DNA ligases showed that the amino acids inmotifs I III IIIa IV V and VI contact the substrates AMPor DNA The important roles of the individual amino acidsin these motifs were verified by alanine scanning mutationalstudies and confirmed as essential for one or more steps ofthe ligation pathway [24] For example motif I (KxDGxR)

Ligase ATP

Ligase - AMP

ppi

Ligase - AMP -

Ligase -

LigaseAMP

Step 1

Step 2

Step 3

P

P

AMPP

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

Figure 1 Schematic diagram of the three-step reaction catalyzed byATP-dependent DNA ligases The three-step reaction catalyzed byDNA ligase (Ligase) results in the serial transfer of AMP (adenosine51015840-monophosphate) to an active site lysine (step 1) and then to the 51015840-PO4end of DNA (step 2) During step 3 the 31015840-OH end of a second

DNA strand attacks the 51015840-PO4 to release AMP and generate the

ligated DNA product

contains the lysine that becomes covalently linked to AMPin the first step of the ligase reaction [25 26] Furthermorethe arginine and lysine side chains in motif VI (RxDK) orientthe PPi leaving group apically relative to the attacking motifI lysine during step 1 enzyme nucleotidylation reaction [2728]

3 Utilization of DNA Ligases in GeneticResearch Technologies

The in vitro manipulation of DNA has been broadly appliedto studies in molecular genetics microbiology immunol-ogy and oncology and also to practical uses in clinicalassays [29]This technologywas developedwithDNA-relatedenzymes which were discovered by research on the molecu-lar mechanisms of the replication and repair of DNA DNAreplication is initiated by DNA primase which synthesizessmall RNA primers that are subsequently elongated by DNApolymerases [30] Based on its primer extension activity aDNA polymerase can be utilized in the polymerase chainreaction (PCR) which amplifies a single copy of DNA byseveral orders of magnitude in a brief in vitro reaction [31]In addition DNA ligase plays a role in joining Okazakifragments during lagging strandmaturation [32] Based on itsactivity to join twoDNA strands in vitro DNA ligase has beencontributing to recombinant DNA technology for exampleDNA cloning [33] In addition DNA ligase is broadly utilizedfor ligase-mediated mutation detection methods and DNAsequencing

31 Oligonucleotide Ligation Assay (OLA) DNA ligase canonly join two DNA strands when they are perfectly

Archaea 3

5774261292350

76

106

290

312

370

102

58

347

379

437

185

246

416

458

518

298

358

561

621

688

I

III

IIIa

IV V

VI

middot middot middot------AITKPLLAATLENIEDVQF------PCLATPKIDGIRSVKQ--T-middot middot middot

middot middot middot----VNIKTNPFKAVSFV-ESAIKKALDNAGYLIAEIKYDGVRGNICVDNTmiddot middot middot

middot middot middotKVQVQLGKPIKPMLAQQAASIRDALLEMGG-EAEFEIKYDGARVQVHKD-Gmiddot middot middot

middot middot middotTLKPQVGIPIRPMLAERLSNPEEILKKMGG-NAIVDYKYDGERAQIHKK-Emiddot middot middot

middot middot middotHCKLSPGIPLKPMLAHPTRGISEVLKRFEEAAFTCEYKYDGQRAQIHALEGmiddot middot middot

middot middot middotEGSDGEISIEGATFQD----

middot middot middotFMLDGELMVKGVDFNTGSGLmiddot middot middot

middot middot middot

middot middot middotAIVEGELVAIG-ENGRPLPFmiddot middot middot

middot middot middotFIIEGEIVAIDPESGEMRPFmiddot middot middot

middot middot middotFILDTEAVAWDREKKQIQPFmiddot middot middot

middot middot middotYWFDYVT-----Dmiddot middot middot

middot middot middotKLYAILPLHIVESmiddot middot middot

middot middot middotNLFDVLYVDGQSLmiddot middot middot

middot middot middotFLFDLMYYEDVDYmiddot middot middot

middot middot middotYAFDLIYLNGESLmiddot middot middot

middot middot middotLLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMK----QFKDAEATmiddot middot middot

middot middot middotLQQLYEQKRAEGHEGLIVKDP--MCIYKR-GKKS-GWWKMK------PENEADmiddot middot middot

middot middot middotAEAFYKRALEMGHEGLMAKRL--DAVYEP-GNRGKKWLKIK-----PTMENLDmiddot middot middot

middot middot middotLKSFFYRAISEGGEGVMVKAIGKDAIYQA-GARGWLWIKLKRDYQSEMADTVDmiddot middot middot

middot middot middotIAEFLEQSVKDSCEGLMVKTLDVDATYEI-AKRSHNWLKLKKDYLDGVGDTLDmiddot middot middot

middot middot middotPRFPVFIGIRHEEDR-----------------------------------

middot middot middotLRHPSFVMFRGTEDNPQEKM------------------------------

middot middot middotPRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES----------

middot middot middotPRFIR-W--RDDKSPEDATTTDEILEMYNKQPKKKIESPAVDESV-----

middot middot middotPRFIR- REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

ChV 22

T7 29Pfu 199

Sso 229

Hu 281

ChV 61

T7 87Pfu 272

Sso 293

Hu 351

ChV 95

T7 46Pfu 336

Sso 367

Hu 246

ChV 134

T7 196Pfu 372

Sso 407

Hu 467

ChV 284

T7 339Pfu 525

Sso 580

Hu 873

Figure 2 Conserved sequence elements define a superfamily of covalent nucleotidyltransferases Six collinear sequence elements designatedas motifs I III IIIa IV V and VI are conserved in capping enzymes and ligases The amino acid sequences are aligned for the DNA ligasesfrom Chlorella virus (ChV) bacteriophage T7 (T7) Pyrococcus furiosus (Pfu) Sulfolobus solfataricus (Sso) and human (Hu) The number ofintervening amino acid residues is indicated Conserved residues are highlighted in red (nucleotidyltransferases) and blue (DNA and RNAligases)

hybridized to a complementary DNA sequence Even asingle base pair mismatch between two strands significantlydecreases the efficiency of the strand joining reaction [34]In 1988 the Oligonucleotide Ligation Assay (OLA) wasdeveloped as a useful method to detect the genotype of thetarget DNA by utilizing this characteristic [6 35] This wasthe first example of the development of a method for geneanalysis by using DNA ligase

OLA consists of two steps First the two probes to behybridized with the target DNA at the sequence of interestmust lie directly adjacent to one another In this case oneprobe must contain a reporter group which is either 32P- orfluorescently labeled and the other must include a recogni-tion group for immobilization such as a biotin group which

could be captured by streptavidin immobilized on a solidsupport Second the neighboring probes that are perfectlyhybridized to the target DNA at the sequence of interestare joined by DNA ligase Then the ligation signal is subse-quently captured by streptavidin and detected by autoradio-graphy or fluorography (Figure 3)This assay takes advantageof the enzymatic accuracy in two events the hybridizationof the probes and the joining by DNA ligase resulting in thereliable analysis of the clinical genotype

A breakthrough in OLA for practical use occurred in1990 when PCRwas coupled to the assay prior to the ligationreaction (PCR-OLA) [36] PCR-OLA amplification only forthe target DNA enhanced the sensitivity of the assay enabl-ing the nonradioactive detection of theOLA results Recently

4 Archaea

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Fluorescence signal No fluorescence signal

Fluorescence detection

Figure 3 Schematic diagrams for the Oligonucleotide Ligation Assay (OLA) DNA fragments from the normal type and the mutant type(at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) Denature samples are heated to 85ndash95∘C forone to several minutes to denature (separate into single strands) the target DNA (2) Hybridization two oligonucleotide probes 51015840-biotinlabeled probes and 31015840-fluorescent (or radiolabeled) probes which are complementary to the normal-type target hybridize to the target DNAfragment (3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase (4) Binding tosupport the fluorescently labeled ligation samples are immobilized to streptavidin and detected by fluorography only if ligated to biotinylatedoligonucleotides that can be bound to streptavidin on a solid support (left)

Archaea 5

Target DNA (wild type)

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(1) Denature

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Probes

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A 3998400

Repeat (1)ndash(3) (n cycle)

Figure 4 Schematic diagrams for the Ligation Detection Reaction (LDR) processes DNA fragments from the normal type and the mutanttype (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) The denaturation process is the same as inFigure 3 (1) (2) Hybridization two oligonucleotide probes complementary to the normal-type target hybridize to the target DNA fragment(3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase and thermocycling linearlyamplifies the amount of product formed In the case of the mutant type template the presence of a single-base mismatch at the junctioninhibits the ligation and therefore no ligated LDR probes are formed (right)

a sensitive specific and high-throughput OLA was devel-oped for the detection of genotypic human immunodefi-ciency virus type 1 (HIV-1) resistance to a Food and DrugAdministration-approved protease [37] This report revealedthat OLA can be used for the detection of the HIV-1 genotype(wild type or mutant) as a genetic diagnosis method

32 DNA Ligase-Mediated Cycling of the Ligation ReactionFurther spreading of DNA ligase-mediated technologies wasachieved by cycling the ligation reaction like PCR using

a thermostable DNA ligase which became available com-mercially in 1990 The thermostable DNA ligase enables thecycling OLA reaction OLA reaction method with thermalcycling procedure developed by using thermostable DNAligase is often specifically termed LigationDetectionReaction(LDR) (Figure 4)Through repeated denaturation annealingand ligation the target signal is amplified linearly [38ndash40]Ligation Chain Reaction (LCR) [38 39 41] and LigationAmplification Reaction (LAR) [42] are also performed byrepeated cycles of heat denaturation of a DNA template

6 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

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T

A

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T GA

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TA

Probes

T

A

TA

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n

Figure 5 Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes (Here wedescribe LCR) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown asreaction templates (1)The denaturation process is the same as in Figure 3 (1) (2) Hybridization four oligonucleotide probes complementaryto the normal-type target hybridize to the target DNA fragment and (3) Ligation adjacent probes that are perfectly complementary to thetarget (left) are connected by DNA ligase Ligated LCR probes from the first round of the ligation become the targets for the subsequent roundusing another probe set (dotted lines) complementary to the first set Thus the amount of ligated LCR probes increases exponentially In thecase of the mutant type template the presence of a single-base mismatch at the junction inhibits the ligation and therefore no ligated LCRprobes are formed (right)

containing the target sequence annealing of probes andligation processes After annealing the first set of two adjacentoligonucleotide probes to the target DNA sequence in aunique manner a second set of complementary oligonu-cleotide probes that hybridize to the sequence opposite to thetarget DNA sequence is applied (Figure 5)Thereafter a ther-mostableDNA ligase will covalently link each pair of adjacent

probes that are completely hybridized at the junction of theadjacent probes Through the repeated sequential processesincluding denaturation annealing and ligation the targetsignal is amplified exponentially These methods require athermostable DNA ligase to allow the ligation to occurunder temperature conditions that prevent mismatches fromhybridizing to form acceptable substrates The thermostable

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

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A C

(2) Hybridization

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Padrock probes

RCA products No RCA products

T

T T

T T

A

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Fluorescent probes

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Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

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Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

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Universal primer (n minus 1)

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Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

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Human (hLigI)

AdDAdD

AdDAdDAdD

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OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

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AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

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OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 3

5774261292350

76

106

290

312

370

102

58

347

379

437

185

246

416

458

518

298

358

561

621

688

I

III

IIIa

IV V

VI

middot middot middot------AITKPLLAATLENIEDVQF------PCLATPKIDGIRSVKQ--T-middot middot middot

middot middot middot----VNIKTNPFKAVSFV-ESAIKKALDNAGYLIAEIKYDGVRGNICVDNTmiddot middot middot

middot middot middotKVQVQLGKPIKPMLAQQAASIRDALLEMGG-EAEFEIKYDGARVQVHKD-Gmiddot middot middot

middot middot middotTLKPQVGIPIRPMLAERLSNPEEILKKMGG-NAIVDYKYDGERAQIHKK-Emiddot middot middot

middot middot middotHCKLSPGIPLKPMLAHPTRGISEVLKRFEEAAFTCEYKYDGQRAQIHALEGmiddot middot middot

middot middot middotEGSDGEISIEGATFQD----

middot middot middotFMLDGELMVKGVDFNTGSGLmiddot middot middot

middot middot middot

middot middot middotAIVEGELVAIG-ENGRPLPFmiddot middot middot

middot middot middotFIIEGEIVAIDPESGEMRPFmiddot middot middot

middot middot middotFILDTEAVAWDREKKQIQPFmiddot middot middot

middot middot middotYWFDYVT-----Dmiddot middot middot

middot middot middotKLYAILPLHIVESmiddot middot middot

middot middot middotNLFDVLYVDGQSLmiddot middot middot

middot middot middotFLFDLMYYEDVDYmiddot middot middot

middot middot middotYAFDLIYLNGESLmiddot middot middot

middot middot middotLLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMK----QFKDAEATmiddot middot middot

middot middot middotLQQLYEQKRAEGHEGLIVKDP--MCIYKR-GKKS-GWWKMK------PENEADmiddot middot middot

middot middot middotAEAFYKRALEMGHEGLMAKRL--DAVYEP-GNRGKKWLKIK-----PTMENLDmiddot middot middot

middot middot middotLKSFFYRAISEGGEGVMVKAIGKDAIYQA-GARGWLWIKLKRDYQSEMADTVDmiddot middot middot

middot middot middotIAEFLEQSVKDSCEGLMVKTLDVDATYEI-AKRSHNWLKLKKDYLDGVGDTLDmiddot middot middot

middot middot middotPRFPVFIGIRHEEDR-----------------------------------

middot middot middotLRHPSFVMFRGTEDNPQEKM------------------------------

middot middot middotPRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES----------

middot middot middotPRFIR-W--RDDKSPEDATTTDEILEMYNKQPKKKIESPAVDESV-----

middot middot middotPRFIR- REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

ChV 22

T7 29Pfu 199

Sso 229

Hu 281

ChV 61

T7 87Pfu 272

Sso 293

Hu 351

ChV 95

T7 46Pfu 336

Sso 367

Hu 246

ChV 134

T7 196Pfu 372

Sso 407

Hu 467

ChV 284

T7 339Pfu 525

Sso 580

Hu 873

Figure 2 Conserved sequence elements define a superfamily of covalent nucleotidyltransferases Six collinear sequence elements designatedas motifs I III IIIa IV V and VI are conserved in capping enzymes and ligases The amino acid sequences are aligned for the DNA ligasesfrom Chlorella virus (ChV) bacteriophage T7 (T7) Pyrococcus furiosus (Pfu) Sulfolobus solfataricus (Sso) and human (Hu) The number ofintervening amino acid residues is indicated Conserved residues are highlighted in red (nucleotidyltransferases) and blue (DNA and RNAligases)

hybridized to a complementary DNA sequence Even asingle base pair mismatch between two strands significantlydecreases the efficiency of the strand joining reaction [34]In 1988 the Oligonucleotide Ligation Assay (OLA) wasdeveloped as a useful method to detect the genotype of thetarget DNA by utilizing this characteristic [6 35] This wasthe first example of the development of a method for geneanalysis by using DNA ligase

OLA consists of two steps First the two probes to behybridized with the target DNA at the sequence of interestmust lie directly adjacent to one another In this case oneprobe must contain a reporter group which is either 32P- orfluorescently labeled and the other must include a recogni-tion group for immobilization such as a biotin group which

could be captured by streptavidin immobilized on a solidsupport Second the neighboring probes that are perfectlyhybridized to the target DNA at the sequence of interestare joined by DNA ligase Then the ligation signal is subse-quently captured by streptavidin and detected by autoradio-graphy or fluorography (Figure 3)This assay takes advantageof the enzymatic accuracy in two events the hybridizationof the probes and the joining by DNA ligase resulting in thereliable analysis of the clinical genotype

A breakthrough in OLA for practical use occurred in1990 when PCRwas coupled to the assay prior to the ligationreaction (PCR-OLA) [36] PCR-OLA amplification only forthe target DNA enhanced the sensitivity of the assay enabl-ing the nonradioactive detection of theOLA results Recently

4 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A A

C

T G

A

A Biotin labeled probes

A

A

Fluorescent probes

B

S

Streptavidin

(4) Binding to support

AS

A

5998400

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5998400

3998400

B

B

BB

BB

Fluorescence signal No fluorescence signal

Fluorescence detection

Figure 3 Schematic diagrams for the Oligonucleotide Ligation Assay (OLA) DNA fragments from the normal type and the mutant type(at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) Denature samples are heated to 85ndash95∘C forone to several minutes to denature (separate into single strands) the target DNA (2) Hybridization two oligonucleotide probes 51015840-biotinlabeled probes and 31015840-fluorescent (or radiolabeled) probes which are complementary to the normal-type target hybridize to the target DNAfragment (3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase (4) Binding tosupport the fluorescently labeled ligation samples are immobilized to streptavidin and detected by fluorography only if ligated to biotinylatedoligonucleotides that can be bound to streptavidin on a solid support (left)

Archaea 5

Target DNA (wild type)

ATarget DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A A

C

T GA

A

Probes

A

A

LDR products

No LDR products

3998400

5998400

5998400

5998400

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3998400

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59984005

998400

3998400

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3998400

3998400 3

9984003998400

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3998400

59984005

998400

3998400

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5998400

3998400

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5998400

5998400

3998400

3998400

3998400

3998400

3998400

[ ]n

A 3998400

Repeat (1)ndash(3) (n cycle)

Figure 4 Schematic diagrams for the Ligation Detection Reaction (LDR) processes DNA fragments from the normal type and the mutanttype (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) The denaturation process is the same as inFigure 3 (1) (2) Hybridization two oligonucleotide probes complementary to the normal-type target hybridize to the target DNA fragment(3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase and thermocycling linearlyamplifies the amount of product formed In the case of the mutant type template the presence of a single-base mismatch at the junctioninhibits the ligation and therefore no ligated LDR probes are formed (right)

a sensitive specific and high-throughput OLA was devel-oped for the detection of genotypic human immunodefi-ciency virus type 1 (HIV-1) resistance to a Food and DrugAdministration-approved protease [37] This report revealedthat OLA can be used for the detection of the HIV-1 genotype(wild type or mutant) as a genetic diagnosis method

32 DNA Ligase-Mediated Cycling of the Ligation ReactionFurther spreading of DNA ligase-mediated technologies wasachieved by cycling the ligation reaction like PCR using

a thermostable DNA ligase which became available com-mercially in 1990 The thermostable DNA ligase enables thecycling OLA reaction OLA reaction method with thermalcycling procedure developed by using thermostable DNAligase is often specifically termed LigationDetectionReaction(LDR) (Figure 4)Through repeated denaturation annealingand ligation the target signal is amplified linearly [38ndash40]Ligation Chain Reaction (LCR) [38 39 41] and LigationAmplification Reaction (LAR) [42] are also performed byrepeated cycles of heat denaturation of a DNA template

6 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A

T

A

T

C

T GA

TA

TA

Probes

T

A

TA

LCR products

No LCR products

5998400

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3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

Repeat (1)ndash(3) (n cycle)

n

Figure 5 Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes (Here wedescribe LCR) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown asreaction templates (1)The denaturation process is the same as in Figure 3 (1) (2) Hybridization four oligonucleotide probes complementaryto the normal-type target hybridize to the target DNA fragment and (3) Ligation adjacent probes that are perfectly complementary to thetarget (left) are connected by DNA ligase Ligated LCR probes from the first round of the ligation become the targets for the subsequent roundusing another probe set (dotted lines) complementary to the first set Thus the amount of ligated LCR probes increases exponentially In thecase of the mutant type template the presence of a single-base mismatch at the junction inhibits the ligation and therefore no ligated LCRprobes are formed (right)

containing the target sequence annealing of probes andligation processes After annealing the first set of two adjacentoligonucleotide probes to the target DNA sequence in aunique manner a second set of complementary oligonu-cleotide probes that hybridize to the sequence opposite to thetarget DNA sequence is applied (Figure 5)Thereafter a ther-mostableDNA ligase will covalently link each pair of adjacent

probes that are completely hybridized at the junction of theadjacent probes Through the repeated sequential processesincluding denaturation annealing and ligation the targetsignal is amplified exponentially These methods require athermostable DNA ligase to allow the ligation to occurunder temperature conditions that prevent mismatches fromhybridizing to form acceptable substrates The thermostable

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

A C

(2) Hybridization

A C

C

Rolling-Circle Amplification(RCA) and detection

A

Padrock probes

RCA products No RCA products

T

T T

T T

A

T T

Fluorescent probes

(1) 5998400-3998400 exonucleolysis

(3) Ligation and5998400-3998400 exonucleolysis

5998400

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39984003

998400

3998400

Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

3998400

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998400

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Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A A

C

T G

A

A Biotin labeled probes

A

A

Fluorescent probes

B

S

Streptavidin

(4) Binding to support

AS

A

5998400

3998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

5998400

3998400

3998400

3998400

5998400

3998400

5998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

B

B

BB

BB

Fluorescence signal No fluorescence signal

Fluorescence detection

Figure 3 Schematic diagrams for the Oligonucleotide Ligation Assay (OLA) DNA fragments from the normal type and the mutant type(at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) Denature samples are heated to 85ndash95∘C forone to several minutes to denature (separate into single strands) the target DNA (2) Hybridization two oligonucleotide probes 51015840-biotinlabeled probes and 31015840-fluorescent (or radiolabeled) probes which are complementary to the normal-type target hybridize to the target DNAfragment (3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase (4) Binding tosupport the fluorescently labeled ligation samples are immobilized to streptavidin and detected by fluorography only if ligated to biotinylatedoligonucleotides that can be bound to streptavidin on a solid support (left)

Archaea 5

Target DNA (wild type)

ATarget DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A A

C

T GA

A

Probes

A

A

LDR products

No LDR products

3998400

5998400

5998400

5998400

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

59984005

998400

3998400

5998400

3998400

3998400 3

9984003998400

5998400

3998400

5998400

3998400

59984005

998400

3998400

5998400

5998400

3998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

3998400

[ ]n

A 3998400

Repeat (1)ndash(3) (n cycle)

Figure 4 Schematic diagrams for the Ligation Detection Reaction (LDR) processes DNA fragments from the normal type and the mutanttype (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) The denaturation process is the same as inFigure 3 (1) (2) Hybridization two oligonucleotide probes complementary to the normal-type target hybridize to the target DNA fragment(3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase and thermocycling linearlyamplifies the amount of product formed In the case of the mutant type template the presence of a single-base mismatch at the junctioninhibits the ligation and therefore no ligated LDR probes are formed (right)

a sensitive specific and high-throughput OLA was devel-oped for the detection of genotypic human immunodefi-ciency virus type 1 (HIV-1) resistance to a Food and DrugAdministration-approved protease [37] This report revealedthat OLA can be used for the detection of the HIV-1 genotype(wild type or mutant) as a genetic diagnosis method

32 DNA Ligase-Mediated Cycling of the Ligation ReactionFurther spreading of DNA ligase-mediated technologies wasachieved by cycling the ligation reaction like PCR using

a thermostable DNA ligase which became available com-mercially in 1990 The thermostable DNA ligase enables thecycling OLA reaction OLA reaction method with thermalcycling procedure developed by using thermostable DNAligase is often specifically termed LigationDetectionReaction(LDR) (Figure 4)Through repeated denaturation annealingand ligation the target signal is amplified linearly [38ndash40]Ligation Chain Reaction (LCR) [38 39 41] and LigationAmplification Reaction (LAR) [42] are also performed byrepeated cycles of heat denaturation of a DNA template

6 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A

T

A

T

C

T GA

TA

TA

Probes

T

A

TA

LCR products

No LCR products

5998400

5998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

5998400

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

5998400

3998400

Repeat (1)ndash(3) (n cycle)

n

Figure 5 Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes (Here wedescribe LCR) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown asreaction templates (1)The denaturation process is the same as in Figure 3 (1) (2) Hybridization four oligonucleotide probes complementaryto the normal-type target hybridize to the target DNA fragment and (3) Ligation adjacent probes that are perfectly complementary to thetarget (left) are connected by DNA ligase Ligated LCR probes from the first round of the ligation become the targets for the subsequent roundusing another probe set (dotted lines) complementary to the first set Thus the amount of ligated LCR probes increases exponentially In thecase of the mutant type template the presence of a single-base mismatch at the junction inhibits the ligation and therefore no ligated LCRprobes are formed (right)

containing the target sequence annealing of probes andligation processes After annealing the first set of two adjacentoligonucleotide probes to the target DNA sequence in aunique manner a second set of complementary oligonu-cleotide probes that hybridize to the sequence opposite to thetarget DNA sequence is applied (Figure 5)Thereafter a ther-mostableDNA ligase will covalently link each pair of adjacent

probes that are completely hybridized at the junction of theadjacent probes Through the repeated sequential processesincluding denaturation annealing and ligation the targetsignal is amplified exponentially These methods require athermostable DNA ligase to allow the ligation to occurunder temperature conditions that prevent mismatches fromhybridizing to form acceptable substrates The thermostable

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

A C

(2) Hybridization

A C

C

Rolling-Circle Amplification(RCA) and detection

A

Padrock probes

RCA products No RCA products

T

T T

T T

A

T T

Fluorescent probes

(1) 5998400-3998400 exonucleolysis

(3) Ligation and5998400-3998400 exonucleolysis

5998400

5998400

3998400

5998400

3998400

3998400

5998400 3

998400

5998400

5998400

3998400

5998400 3

998400

5998400

5998400

5998400

3998400

5998400

5998400

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

39984003

998400

3998400

Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

3998400

3998400

3998400

3998400

3998400

3998400

3998400

3998400

5998400

5998400

5998400 5

998400

5998400

5998400

5998400

5998400

Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

5998400

5998400

5998400

3998400

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3998400

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3998400

3998400

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3998400

3998400

3998400

3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 5

Target DNA (wild type)

ATarget DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A A

C

T GA

A

Probes

A

A

LDR products

No LDR products

3998400

5998400

5998400

5998400

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

5998400

5998400

3998400

3998400

5998400

3998400

5998400

59984005

998400

3998400

5998400

3998400

3998400 3

9984003998400

5998400

3998400

5998400

3998400

59984005

998400

3998400

5998400

5998400

3998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

3998400

[ ]n

A 3998400

Repeat (1)ndash(3) (n cycle)

Figure 4 Schematic diagrams for the Ligation Detection Reaction (LDR) processes DNA fragments from the normal type and the mutanttype (at the single nucleotide polymorphism (SNP) site) are shown as reaction templates (1) The denaturation process is the same as inFigure 3 (1) (2) Hybridization two oligonucleotide probes complementary to the normal-type target hybridize to the target DNA fragment(3) Ligation adjacent probes that are perfectly complementary to the target (left) are connected by DNA ligase and thermocycling linearlyamplifies the amount of product formed In the case of the mutant type template the presence of a single-base mismatch at the junctioninhibits the ligation and therefore no ligated LDR probes are formed (right)

a sensitive specific and high-throughput OLA was devel-oped for the detection of genotypic human immunodefi-ciency virus type 1 (HIV-1) resistance to a Food and DrugAdministration-approved protease [37] This report revealedthat OLA can be used for the detection of the HIV-1 genotype(wild type or mutant) as a genetic diagnosis method

32 DNA Ligase-Mediated Cycling of the Ligation ReactionFurther spreading of DNA ligase-mediated technologies wasachieved by cycling the ligation reaction like PCR using

a thermostable DNA ligase which became available com-mercially in 1990 The thermostable DNA ligase enables thecycling OLA reaction OLA reaction method with thermalcycling procedure developed by using thermostable DNAligase is often specifically termed LigationDetectionReaction(LDR) (Figure 4)Through repeated denaturation annealingand ligation the target signal is amplified linearly [38ndash40]Ligation Chain Reaction (LCR) [38 39 41] and LigationAmplification Reaction (LAR) [42] are also performed byrepeated cycles of heat denaturation of a DNA template

6 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A

T

A

T

C

T GA

TA

TA

Probes

T

A

TA

LCR products

No LCR products

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Repeat (1)ndash(3) (n cycle)

n

Figure 5 Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes (Here wedescribe LCR) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown asreaction templates (1)The denaturation process is the same as in Figure 3 (1) (2) Hybridization four oligonucleotide probes complementaryto the normal-type target hybridize to the target DNA fragment and (3) Ligation adjacent probes that are perfectly complementary to thetarget (left) are connected by DNA ligase Ligated LCR probes from the first round of the ligation become the targets for the subsequent roundusing another probe set (dotted lines) complementary to the first set Thus the amount of ligated LCR probes increases exponentially In thecase of the mutant type template the presence of a single-base mismatch at the junction inhibits the ligation and therefore no ligated LCRprobes are formed (right)

containing the target sequence annealing of probes andligation processes After annealing the first set of two adjacentoligonucleotide probes to the target DNA sequence in aunique manner a second set of complementary oligonu-cleotide probes that hybridize to the sequence opposite to thetarget DNA sequence is applied (Figure 5)Thereafter a ther-mostableDNA ligase will covalently link each pair of adjacent

probes that are completely hybridized at the junction of theadjacent probes Through the repeated sequential processesincluding denaturation annealing and ligation the targetsignal is amplified exponentially These methods require athermostable DNA ligase to allow the ligation to occurunder temperature conditions that prevent mismatches fromhybridizing to form acceptable substrates The thermostable

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

A C

(2) Hybridization

A C

C

Rolling-Circle Amplification(RCA) and detection

A

Padrock probes

RCA products No RCA products

T

T T

T T

A

T T

Fluorescent probes

(1) 5998400-3998400 exonucleolysis

(3) Ligation and5998400-3998400 exonucleolysis

5998400

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Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

3998400

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5998400 5

998400

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Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

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TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 Archaea

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

(1) Denature

A C

T G

(2) Hybridization

A C

T G

(3) Ligation

A

T

A

T

C

T GA

TA

TA

Probes

T

A

TA

LCR products

No LCR products

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Repeat (1)ndash(3) (n cycle)

n

Figure 5 Schematic diagrams for the Ligation Chain Reaction (LCR) and Ligation Amplification Reaction (LAR) processes (Here wedescribe LCR) DNA fragments from the normal type and the mutant type (at the single nucleotide polymorphism (SNP) site) are shown asreaction templates (1)The denaturation process is the same as in Figure 3 (1) (2) Hybridization four oligonucleotide probes complementaryto the normal-type target hybridize to the target DNA fragment and (3) Ligation adjacent probes that are perfectly complementary to thetarget (left) are connected by DNA ligase Ligated LCR probes from the first round of the ligation become the targets for the subsequent roundusing another probe set (dotted lines) complementary to the first set Thus the amount of ligated LCR probes increases exponentially In thecase of the mutant type template the presence of a single-base mismatch at the junction inhibits the ligation and therefore no ligated LCRprobes are formed (right)

containing the target sequence annealing of probes andligation processes After annealing the first set of two adjacentoligonucleotide probes to the target DNA sequence in aunique manner a second set of complementary oligonu-cleotide probes that hybridize to the sequence opposite to thetarget DNA sequence is applied (Figure 5)Thereafter a ther-mostableDNA ligase will covalently link each pair of adjacent

probes that are completely hybridized at the junction of theadjacent probes Through the repeated sequential processesincluding denaturation annealing and ligation the targetsignal is amplified exponentially These methods require athermostable DNA ligase to allow the ligation to occurunder temperature conditions that prevent mismatches fromhybridizing to form acceptable substrates The thermostable

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

A C

(2) Hybridization

A C

C

Rolling-Circle Amplification(RCA) and detection

A

Padrock probes

RCA products No RCA products

T

T T

T T

A

T T

Fluorescent probes

(1) 5998400-3998400 exonucleolysis

(3) Ligation and5998400-3998400 exonucleolysis

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Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

3998400

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Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

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TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 7

DNA ligase made this assay useful for DNA-based diag-nostic tests for inherited diseases in clinical laboratoriesThe ligation-mediated detection of point mutations by ther-mostable DNA ligase is now used to detect hepatitis Bvirus (HBV) mutants [43ndash45] colon tumor microsatellitesequence alterations [46] the mutation responsible forbovine leukocyte adhesion deficiency [47] AZT-resistanceHIV mutants [48] mutations in the ras family of oncogenes[49] extended spectrum 120573-lactamase resistance in bacteria[50] and ganciclovir-resistant cytomegalovirus mutants [51]

33 Padlock Probe and Rolling-Circle Amplification (RCA)Padlock probes and RCA are alternative methods for detect-ing known sequence variants in DNA and particularly fordetecting single nucleotide polymorphisms by using a ther-mostable DNA ligase The padlock probe has target recogni-tion sequences situated at both the 51015840- and 31015840-ends connectedby an intervening sequence that can include the sequence fordetectionWhen the probe is hybridized to a target sequencethe two ends are brought adjacent to each other and thena thermostable DNA ligase covalently joins the two endsand circularizes the probe This circularized probe is woundaround the target strand in a manner similar to a padlockdriven by the helical nature of the double-stranded DNA[52] (Figure 6) This probe is used for the localization ofsignals in in situ analyses For example a padlock probewas able to detect repeated alphoid sequences in metaphasechromosomes in a living cell demonstrating its utility forin situ studies [53ndash55] RCA is also known as a simple andefficient isothermal enzymatic process that utilizes strand-displacement DNA and RNA polymerases like Phi29 Bstand Vent exo-DNA polymerases for DNA and T7 RNApolymerase for RNA to generate long single stranded DNAsand RNAs containing tens to hundreds of tandem repeatsthat are complementary to the circular template [56ndash58]The padlock principle has been combined with RCA formutation detection in which a primer annealing to the linkerregion initiates rolling circle replication (Figure 6) becausethe padlock approach alone is not sufficient to detect singlenucleotide differences in a single copy gene in situ [59ndash61] Point mutations in the cystic fibrosis transmembraneconductance regulator (CFTR) p53 BRCA1 and Gorlinsyndrome genes were visualized in interphase nuclei andDNA fibers by RCA [62] These results demonstrated thatligase-mediated mutation detection methods coupled withRCA are able to reveal single nucleotide differences in a singlecell The ability to detect mutations in a cellular milieu hasimportant implications for cancer research and diagnosis

In another aspect of the RCA with ligation methodsProximity Ligation Assay (PLA) is known for one of thepotent techniques for detecting individual proteins or proteincomplexes in situ by using antibodies with attached DNAstrands that participate in ligation and subsequent RCAreactions [63] PLA can be used for detecting reactions inwhich identification of target molecules depends on tworecognition events Formation of a proper target complexresults in the formation of a circular DNA strand by exoge-nously added DNA ligase This circular DNA is used totemplate a locally restricted RCA reaction which generates

Target DNA (wild type)

A

Target DNA (mutant)

C

T G

A C

(2) Hybridization

A C

C

Rolling-Circle Amplification(RCA) and detection

A

Padrock probes

RCA products No RCA products

T

T T

T T

A

T T

Fluorescent probes

(1) 5998400-3998400 exonucleolysis

(3) Ligation and5998400-3998400 exonucleolysis

5998400

5998400

3998400

5998400

3998400

3998400

5998400 3

998400

5998400

5998400

3998400

5998400 3

998400

5998400

5998400

5998400

3998400

5998400

5998400

5998400

5998400

5998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

39984003

998400

3998400

Figure 6 Schematic representation of the target-primed Rolling-Circle Amplification (RCA) of circularized padlock probes (1) 51015840ndash31015840exonucleolysis the target DNA is restriction digested 31015840 of the targetsequence and irreversibly made single stranded by strand-specific51015840ndash31015840 exonucleolysis (2) Hybridization padlock probes (red) withthe tag sequence (green) are hybridized to their target sequences(3) Ligation amp 51015840ndash31015840 exonucleolysis adjacent padlock probes thatare perfectly complementary to the target (left) are connectedby DNA ligase thus locking the probe onto the target moleculeAfter ligation the RCA is initiated by the Phi29 DNA polymeraseby turning the target molecule into a primer through the 31015840ndash51015840exonucleolysis of any 31015840 end protruding beyond the padlock probehybridization site The padlock probe then serves as the templatefor DNA synthesis The RCA product (black) is detected throughthe hybridization of fluorescently labeled probes (green) to tagsequences specific for the padlock probe Arrowheads indicate 31015840ends of the RCA reaction

an elongating ssDNA rolling-up in a ball that can be detectedwhen hybridized with fluorescence-labeled probes [7] Thebinding to a target interacting complex by two different anti-bodies with attached oligonucleotides individually (referredto as proximity probes) is followed by the addition of twomore oligonucleotides that are then ligated into a circular

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

3998400

3998400

3998400

3998400

3998400

3998400

3998400

3998400

5998400

5998400

5998400 5

998400

5998400

5998400

5998400

5998400

Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

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3998400

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3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

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International Journal of

Volume 2014

Zoology

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GenomicsInternational Journal of

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BioinformaticsAdvances in

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Enzyme Research

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International Journal of

Microbiology

8 Archaea

(1) Protein interaction

(2) Proximity probe binding

Proximity probes

(3) Hybridize connector oligoConnector oligo

(4) Ligation to form complete DNA circle

(5) Rolling circle amplification (RCA) and detection Fluorescent probes

3998400

3998400

3998400

3998400

3998400

3998400

3998400

3998400

5998400

5998400

5998400 5

998400

5998400

5998400

5998400

5998400

Figure 7 Schematic representation of in situ Proximity Ligation Assay (PLA) (1) Protein interaction the complex is formed between targetproteins (light blue and pink) (2) Proximity probe binding antibodies (black) with individual proximity probes (glay) bind to each ofthe target protein complexes (3) Hybridization of connector oligo-DNAs target protein complex serves to template the hybridization ofconnector oligo-DNAs (red) (4) Ligation to form complete DNA circle adjacent connector oligos that are perfectly complementary to thetarget template are connected by DNA ligase (5) Rolling Circle Amplification (RCA) and detection after ligation the RCA is initiated by thePhi29 DNA polymerase by turning the target molecule primed by one of the proximity probes The RCA product is detected through thehybridization of fluorescently labeled probes (green) Arrowheads indicate 31015840 ends and roundheadsmeans 31015840 endsmodified with 21015840 O-methylRNA

DNA strand by the function of DNA ligase The circularDNA strand is templated by the oligonucleotides attachedto antibodies (Figure 7) Next one of the antibody-boundoligonucleotides is utilized as a primer of the succeedingRCA reaction resulting in the generation of an ssDNArolling circle productThe rolling circle is covalently attached

to one of the proximity probes A 60min RCA by Phi29DNA polymerase results in a 1000-fold amplification ofthe 100 nt DNA circle producing an around 100 kb rollingcircle product [63] The rolling circle product is then visual-ized by hybridization of fluorescence-labeled complementaryoligonucleotide detection probes in situ

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 9

34 Next-Generation DNA Sequencing by Using DNA LigaseRecently Next Generation Sequencing (NGS) technologiessuch as the 454 FLX pyrosequencer (Roche) [64] Illuminagenome analyzer (Illumina) [65] and Sequence by Oligonu-cleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) [66] have revolutionized genomic and geneticresearch Although these platforms are quite diverse in termsof their sequencing biochemistry and sequence detectiontheir workflows are conceptually similar to each other Thearray is prepared by random fragmentation of DNA fol-lowed by in vitro ligation of common adaptor sequences byDNA ligase DNA fragments with adapters are amplified byDNA polymerase and are detected by each platform Thedetection platforms rely on sequencing by DNA syntheticmethods that is the serial extension of primed templatesThe enzyme driving the DNA synthesis can be either a DNApolymerase or a DNA ligase The 454 FLX Pyrosequencerand Illumina analyzer use DNA polymerase methods withpyrosequencing [67] and reversible dye terminator technol-ogy respectively [68] In contrast SOLiD uses DNA ligaseand a unique approach to sequence the amplified fragments[69] A universal primer complementary to the adaptorsequence is hybridized to the array of amplicons Each cycle ofsequencing involves the ligation of a degenerate fluorescentlylabeled 8-mer probe set (Figure 8) The octamer mixture isstructured and the type of nucleotide (A T G or C) at thespecific position within the 8-mer probe set correlates withthe type of fluorescent label After the ligation of the 8-merprobes images are acquired in four channels resulting in theeffective collection of sequencing data for the 31015840 end positionsof the probes across all template-bearing beads Then theoctamer is chemically cleaved between positions 3 and 4 toremove the fluorescent label Progressive rounds of octamerligation enable the sequencing of every 5th base (eg bases1 6 11 and 16 of the template strand) After several cyclesof probe ligation reactions the extended primer is denaturedto reset the system Subsequent iterations of this process canbe applied to a different set of positions (eg bases 0 5 10and 15 of the template strand) by using different mixtures ofoctamers in which the nucleotides at the different positionare correlated with the label colors An additional feature ofthis platform involves the use of two-base encoding an error-correction scheme in which two adjacent bases rather than asingle base are correlated with the label Each base positionis then queried twice (in a set of 2 bp interrogated in a givencycle) and thus miscalls can be more readily identified [70]

4 Structural Transition of the DNA Ligase inthe Reaction Sequence

In Section 2 we showed that the sequence alignments clearlyrevealed several homologous regions among ATP-dependentDNA ligases and RNA capping enzymes indicating thepresence of five conserved motifs (I III IIIa IV and V)in these proteins This fact suggests that the nucleotidyl-transferase superfamily members including ATP-dependentDNA ligases may share a similar protein fold and a commonreaction mechanism Next we will present a series of crystalstructures of ATP-dependent DNA ligases and describe the

structural and functional insights into the mechanisms of theenzymes

41 Structural Studies of ATP-Dependent DNA Ligase ATP-dependent DNA ligases show considerable variation in theirmolecular sizes which range from 41 kDa (bacteriophageT7) [71] to 102 kDa (human DNA ligase I (hLigI)) [72]Despite this wide size variation it is clear from the sequencealignments that the smaller enzymes constitute a commoncore structure that is conserved across all ATP-dependentligases [73] The crystal structures of the ATP-dependentDNA ligases such as bacteriophage T7 DNA ligase (T7Lig)complexed with ATP [8 74] and Chlorella virus DNA ligase(ChvLig) with covalently bound AMP [75] have been solved(Figure 9(a) top)These DNA ligases adopt a common archi-tecture of two distinct domains the adenylylation domain(AdD) and the oligonucleotideoligosaccharide-binding-folddomain (OBD) which are jointly called the catalytic coredomains The catalytic core domains are the minimal entityfor the nick-joining activity as seen in the ChvLig and T7Ligenzymes [8 75]TheAdD contains the catalytic lysine residuethat forms a covalent bond with AMP which is derived fromthe substrate ATP The Lys238 and Lys240 residues of T7Lig(Lys188 and Lys186 of ChvLig) within the AdD are essentialfor the adenylylation and nick sealing activities [76 77] TheLys240 residue forms a photo-crosslinking adduct with the51015840-terminal nucleotide of the nick implying its direct involve-ment in binding the phosphate of the nick [78] The OBD isconnected to the AdD via the conserved motif V [5 79] andis similar to other DNA and RNA binding proteins [80 81]The OBD is observed in the related nucleotidyltransferasethe mRNA capping enzyme from Chlorella virus whichundergoes opened-closed conformational changes duringcatalysis The OBD domain of the enzyme was found tomove towards AdD and close the nucleotide-binding pocket[80 81]

In contrast three crystal structures of large ATP-dependent DNA ligases which mainly ligate the nicksbetween Okazaki fragment in DNA replication in Eukaryaand Archaea with an additional N-terminal DNA-bindingdomain (DBD) have been reported (Figure 9(a) bottom) [9ndash11 82 83]This extra domain is not essential for hLigI activityin vitro [84 85] but is considered to be crucial for detecting asingly nicked dsDNA [9] The structures of the two archaealDNA ligases fromPyrococcus furiosus (PfuLig) and Sulfolobussolfataricus (SsoLig) were determined in the closed [11] andextended forms [10] respectively The structure of hLigI incomplex with DNA [9] revealed that the enzyme entirelyencircles the nicked-DNA All of these DNA ligases arecommonly composed of three domains (DBD AdD andOBD) in the sequences from theN toC termini Although theprotein folding of each domain is strikingly similar among thethree DNA ligases the relative domain orientations withineach enzyme are quite different

42 Structural Transition of DNA Ligase Molecules in EachReaction Step Based on the crystal structures describedabove a ligation mechanism was proposed as schematicallyrepresented in Figure 9(b) In solution a DNA ligase partly

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 Archaea

(i) Universal primer (n) hybridization

(vii) Repeat reset with n minus 2 n minus 3 n minus 4 universal primers

(n minus 1) hybridization

Universal primer (n minus 1)

Universal primer (n)

(1) Library preparation

(2) Emulsion PCR

(3) Bead deposition

dsDNADigestion and adapter ligation

Magnetic beads

Glass slide

(4) Sequencing by ligationAdapter

Adapter with ssDNA

Template sequence

TGnnnzzz

TCnnnzzz

TAnnnzzz

TTnnnzzz

(ii) Specific de-base probe ligationand fluorescence detection

Fluoresence

(iii) Cleave off fluor

Fluorescently labeled di-base probes

(iv) Repeat steps (i)ndash(iv)

Adapter

(v) Probe reset and universal primer

with probes

5998400

5998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

3998400

5998400

3998400

5998400

3998400

3998400

3998400

3998400

TTnnnzzzAA

TGnnn TCnnn TAnnnTTnnn

zzz

AA AC AG AT

TTnnnAA

(vi) Repeat steps (i)ndash(iv) with new probe

Figure 8 The ligase-mediated sequencing approach of the Sequence by Oligonucleotide Ligation and Detection (SOLiD) sequencer (LifeTechnologies) (1) Library preparation two different adapters are ligated to sheared genomic DNA (2) Emulsion PCR emulsion PCR isconducted using magnetic beads to generate ldquobead clonesrdquo in which each contains a single nucleic acid species (3) Bead deposition thebeads are then attached to the surface of a glass slide (4) Sequencing by ligation ligase-mediated sequencing begins by annealing a universalprimer to the shared adapter sequences on each amplified fragment (i) and then DNA ligase is provided along with specific fluorescentlylabeled 8-mers in which the two bases at the 31015840 end of the probe are encoded by the attached fluorescent group Each ligation step is followedby fluorescence detection (ii) after which a regeneration step removes the bases from the ligated 8-mer (including the fluorescent group)(iii) and concomitantly prepares the extended probe for another round of ligation (ivndashvii) Since each fluorescent group on a ligated 8-meridentifies a two-base combination the resulting sequence reads can be screened for base-calling errors versus either true polymorphisms orsingle base deletions by aligning the individual reads to a known high-quality reference sequence

adopts the extended form as expected from the SsoLig crystalstructure and the small angle X-ray scattering (SAXS) analy-sis [10] In the absence ofDNAandAMP theOBDof SsoLig isturned away from the AdD in an open conformation resem-bling that seen in the crystal structures of the compact andtwo-domain (AdD-OBD) DNA ligases from Chlorella virusandT7 (Figure 9(a))The closed conformation of the catalyticcore domains which represents the active conformation for

step 1 is observed in the crystal structures of PfuLig asproposed previously [10 11] In step 2 tight binding betweenthe enzyme and the substrate DNA occurs as revealed by thehLigIndashDNA complex crystal structure [9] These structuresdepict three different phosphoryl transfer reactions and theflexible multidomain structure of DNA ligases facilitatesthe adoption of different enzyme conformations during thecourse of the reaction (Figure 9(b)) [10] A superimposition

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

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International Journal of

Volume 2014

Zoology

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Enzyme Research

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International Journal of

Microbiology

Archaea 11

Chlorella virus Bacteriophage T7

Human (hLigI)

AdDAdD

AdDAdDAdD

OBD OBD

OBD

OBD

OBD

Sulfolobus solfataricus (SsoLig)Pyrococcus furiosus (PfuLig)

(a)

DBD

OBD

P

P

AdD OBD

DBD

Step 2

Step 3

Step 1

AdD

AMP

AMP

hLigI PfuLig

AdD

DBD

SsoLig

OBD

AMPAMP

(b)

Figure 9 Crystal structures of ATP-dependentDNA ligases (a)The viral and bacterial ATP-dependentDNA ligases fromChlorella virus (leftblue) and bacteriophage T7 (right yellow) are two-domain ligases These ligases revealed the common structures of two distinct domainsdesignated as the adenylylation domain (AdD) and the oligonucleotideoligosaccharide-binding-fold domain (OBD) which are jointly calledthe catalytic core domains In these two structures the catalytic core domains adopted an open form The three-domain structure of DNAligase is characteristic of the archaeal (Sulfolobus solfataricus SsoLig (orange left) and Pyrococcus furiosus PfuLig (blue right)) and eukaryotic(human hLigI (green center)) DNA ligases The AdD contains most of the catalytic residues and is assisted by two flanking domains thatalso bind to DNA the N-terminal DNA-binding domain (DBD) and the C-terminal OBD Each C-terminal helix is highlighted in yellow(SsoLig) magenta (hLigI) and red (PfuLig) The figure was prepared using Chimera [105] (b) Schematic diagram of the structure-basedligationmechanism Before and after the ligation reaction DNA ligase adopts the extended conformation as expected from the SsoLig crystalstructure In step 1 the closed conformation of the catalytic core domains at the carboxyl terminus in PfuLig creates a small compartmentwhich holds a covalently bound AMPmolecule In step 2 the interactions with the substrate DNAmust be stabilized for the enzyme to adopta compact conformation as seen in the crystal structure of hLig1 bound to the nicked-DNA Finally the ligation of the DNA strands and thesubsequent release of AMP and DNA strands from DNA ligase occur during step 3

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

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Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

12 Archaea

AdD

OBD (Pfu)

OBD (human)

DBD (Pfu)

OBD (Sso)

(a)

Motif VI

hLigI PfuLig

Substrate DNA

(b)

Figure 10 Superimposed ribbon diagrams (a) Ribbon diagrams of PfuLig (2CFM blue) hLigI (1X9N green) and SsoLig (2HIV orange)in which the AdD from each ligase were maximally overlapped with each other The DBD from hLigI and SsoLig were omitted for clarityThe arrangements of the OBD relative to the AdD in each ligase are apparently different from each other Notably the OBD from PfuLig isclosely bound to AdD and is replaced by the bound-DNA substrate (grey) in hLigI (b) Superimposed diagrams of adenylation domains fromPfuLig and hLigI flanked by surface representations of motif VI (PfuLig) and the upstream region of the substrate DNA (hLigI) in whichthe electrostatic distributions (positive charge blue negative charge red) are mapped onto the molecular surfaces Since the electrostaticdistributions on the surfaces nearby the AdD of motif VI and the DNA are both negatively charged the replacement of motif VI with DNAmay easily occur

of the AdDs in PfuLig SsoLig and hLigI revealed that thearrangements of the OBD relative to the AdD in each ligaseare apparently different from each other Notably the OBDfrom PfuLig is closely bound to the AdD and is replacedby the bound-DNA substrate in hLigI (Figure 10(a)) Ribbondiagrams of the AdDs from hLigI and PfuLig together withthe adjacent surface representations of the substrate DNA(hLigI) and motif VI (PfuLig) are shown in Figure 10(b)The electrostatic distribution on the motif VI surface facingAdD is negative suggesting that motif VI is a molecularmimic of the incoming DNA substrate [11] This findingindicates that the ligation could be completed simply bythe smooth replacement between the substrate DNA andmotif VI Indeed in the closed structure of PfuLig motif VI

approaches the active site in the absence of DNA whereas itoccupies a position in the region upstream from the nickedDNA in the structure of hLigI

43 Interface of the Mandatory Domains (AdD and OBD) forthe Enzymatic Reaction In the ATP-dependent DNA ligasesthe enzyme captures ATP to adenylylate a Lys residue in thefirst step in which motif VI is also indispensable [27] MotifVI in PfuLig provides several basic residues located aroundthe AMP binding pocket This electron density contributesto forming the compartment that traps the AMP molecule(Figure 11) In particular R531 and K534 in motif VI specificto the DNA ligases in Archaea and Eukarya contribute to thebasic surface within the pocket (Figure 11) [11]

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 13

K534

R531

AMP

AMPMotif VI

Exit

Exit

90∘

Figure 11 Top the AMP-binding pocket viewed from the insideof the molecule A solvent-accessible surface was created withoutthe bound AMP and then the final refined AMP model wassuperimposed The noncovalently bound AMP is trapped within aclosed compartment which conforms well to the shape of the AMPmolecule Bottom surface representation around the hole in theactive site pocketThe surface ofmotif VI is colored cyanThe boundAMP is depicted as a sphere model The phosphate (phosphorusorange oxygen red) group is visible through a small ldquoexitrdquo

The electrostatic potential distributions of the AdD-OBDinteracting surfaces (Figure 12) revealed that the predom-inant charge distribution on each surface was oppositelycharged and this may be involved in stabilizing the closedconformation of the two catalytic core domains [11] There isa small exit of the pocket through the closed conformationof the two domains (Figure 11) and the diameter of the exitis about 55 A and could accommodate the PPi molecule butnot the AMP moiety The exit may serve for the spontaneousrelease of the PPi product This notion is consistent with thefact that the DNA ligase from Pyrococcus horikoshii whichis more than 90 identical to PfuLig cannot use NAD+ asa nucleotide cofactor [86] because the pocket is too smallto accommodate the nicotinamide ring to be released fromthe active site Presumably this compact ldquoreaction roomrdquo ofPfuLig would have been specifically designed to completethe conversion process from ATP to AMP within the closedpocket [11]

44 Role of the C-Terminal Helix Commonly Observed in theArchaeal and Eukaryotic DNA Ligases The overall architec-tures of the three domains in hLigI and PfuLig are similarto each other (Figure 9(a)) although the sequence identitiesbetween the corresponding fragments of hLigI and PfuLigare moderate (32) The crystal structures and sequenceanalyses revealed that the eukaryotic and archaeal DNAligases possess a C-terminal helix shortly after motif VI(Figures 9(a) and 13(a)) The sequence extension harboringthe C-terminal helix is conserved among the eukaryoticand archaeal DNA ligases whereas the ligases from viruses(Chlorella virus (ChV)) and bacteriophages (bacteriophageT7 (T7)) lack this long extension (Figure 13(a)) The C-terminal helix is located at the boundary of the AdD andthe OBD in the closed structure of PfuLig [11] In the caseof hLigI this helix is distant from the domain interfacebecause of the bound DNA substrate (Figure 9(a)) In facta structural comparison between the DNA-hLigI complexand PfuLig suggested that the C-terminal helix might playa crucial role in switching from the tightly closed formto the DNA-substrate-bound form The C-terminal helix ofPfuLig connects the AdD and the OBD through five polaror ionic interactions (Figure 13(b)) This feature suggests thatthe helix might play a critical role in stabilizing the closedconformation of the catalytic core domains during step 1

5 Engineered DNA Ligases withImproved Abilities

As described in Section 3 DNA ligases are critical for manyapplications in molecular biology Improvements in theenzymatic ability such as the nick sealing efficiency or fidelitywill provide better performance in the current methods forligase-mediated mutation detection and NGS as describedabove Numerous improvements of DNA polymerases havebeen reported [87ndash89] whereas fewer are known for DNAligases Here we describe some reports on improvements ofthe enzymatic performances of DNA ligases

51 Improvement of the Nick-Sealing Activity by Fusion withthe Archaeal DNA Binding Domain The ATP-dependentDNA ligase from bacteriophage T4 (T4Lig) has evolved to bea nick-sealing enzyme It can also join double-stranded DNA(dsDNA) fragments with cohesive ends [90] Furthermoreit is the only commercially available DNA ligase that canjoin blunt-ended DNA duplexes in vitro in the absenceof macromolecular enhancers such as polyethylene glycol[91 92] The ligation reaction of cohesive or blunt-endeddsDNA fragments is prerequisite for NGS which requiresthe in vitro ligation of common adaptor sequences Howeverthe turnover numbers for the fragment joining reactionsof T4Lig are lower than those for nick sealing and T4Ligis approximately five orders of magnitude less efficient injoining blunt-ended duplexes than nick-sealing [92 93]T4Lig is also inefficient for ligating fragments with single baseoverhangs [94] The poor fragment joining activity of T4Ligoften leads to NGS failures In order to improve the efficiencyof fragment joining by T4Lig Wilson et al constructed avariety of chimeras of T4Lig fused with the DNA-binding

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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GenomicsInternational Journal of

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BioinformaticsAdvances in

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Signal TransductionJournal of

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Evolutionary BiologyInternational Journal of

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Advances in

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Nucleic AcidsJournal of

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

14 Archaea

R414

D540AMP

90∘

90∘

Figure 12The electrostatic distributions on the interacting surfaces of AdD (bottom left) andOBD (bottom right) in which one of the ionicinteraction pairs (Arg414 and Asp540) is highlighted as sphere models

domains from other enzymes [92] This mutational strategywas inspired by a previous report in which the geneticfusion of a sequence of a nonspecific archaeal DNA bindingprotein (Sso7d from Sulfolobus solfataricus) to Pfu DNApolymerases resulted in considerably increased processivityand improved performance in polymerase chain reaction(PCR) amplifications [88] The fusion of T4Lig with Sso7dshowed a 60 increase in performance over T4Lig in thecloning of a blunt-ended fragment [92]

52 Improvement of the Fidelity of Thermostable DNA Ligaseby Site-Directed Mutagenesis The specificity of DNA ligaseis exploited in LCR and LDR analyses to distinguish singlebase mutations associated with genetic diseases The ligationfidelity of T4Lig is improved by the presence of spermidineand by high salt and low ligase concentrations [6 34]However the increased fidelity of T4Lig by adjusting thereaction conditions is not sufficient for the ideal detectionof SNPs [6 34 95 96] The thermostable DNA ligases from

Thermus aquaticus (Taq) and Thermus thermophilus (Tth)exhibited far greater fidelity than that reported for T4Lig[39 97]The ligation reactions at the higher temperature pre-ventedmismatched hybridizations in the substrates Luo et alreported the further improvement of the fidelity of TthLig bysite-directed mutagenesis The fidelity of DNA polymeraseswas decreased by site-directed mutagenesis at the motifassociated with primer-template binding or the exoIII motif[98ndash100] However the exoIII motif mutants also knownas ldquoantimutatorrdquo strains showed increased fidelity in whichthe balanced activities of the polymerizing and 3

1015840

rarr 51015840

exonuclease reactions might improve the overall fidelity [99ndash102] Two mutant ligases K294R and K294P were identifiedwith fidelities increased by sim4-fold and 11-fold respectivelybesides retaining their nick sealing activities [97]

53 Structure-Based Mutational Study of an Archaeal DNALigase towards Improvement of the Ligation Activity Asmen-tioned in the previous section thermostable DNA ligases are

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

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Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 15

ChV 283

T7 339

Pfu 525

Tko 528

hu1 873

Sc1 723

middot middot middot PRFPVFIGIRHEEDR

middot middot middot LRHPSFVMFRGTEDNPQEKM

middot middot middot PRFVA-L--RDDKGPEDADTIERIAQLYELQEKMKGKVES

middot middot middot PRYVA-L--REDKSPEEADTIERVAELYELQERFKAKK

middot middot middot PRFIR-V--REDKQPEQATTSAQVACLYRKQSQIQNQQGEDSGSDPEDTY

middot middot middot PRFLR-I--REDKGVEDATSSDQIVELYENQSHMQN

Motif VI

(a)

D540

AdD OBD AdD OBD

D540R414R414

D235D235

K554K554

K558K558

R544R544

Q228Q228Q547Q547

Q229Q229

(b)

Figure 13 Roles of the C-terminal extension helix (a) Alignment of the sequences of motif VI and the C-terminal extension The sequencesof the DNA ligases from a virus (ChV Chlorella virus) bacteriophage (T7 bacteriophage T7) Archaea (Pfu Pyrococcus furiosus TkoThermococcus kodakaraensis) human (hu1 human DNA ligase I) and yeast (Sc1 Saccharomyces cerevisiae DNA ligase I) are aligned Theregion formotif VI is shaded and the extension helix region determined by the crystal structures of PfuLig and hLigI is colored red (residuesafter Q902 of hLigI were not included in the previously reported crystal structure and are colored gray) (b) Stereo diagram of the interfaceof the AdD and the OBD in PfuLig Ball-and-stick models represent the amino acid residues involved in the interaction between the twodomains basic acidic and polar residues are colored blue red and purple respectively The C-terminal helix is colored redThe dotted linesindicate the polar or ionic interactions between AdD and OBD

utilized in LCR and LDR which require a heat denaturationstep However thermostable DNA ligases possess weakerligation activity at lower temperatures (20ndash40∘C) resultingin the decreased efficiency of the LCR and LDR proceduresusing short probes with low-melting temperatures Herewe present a structure-guided mutational analysis of thehyperthermostable DNA ligase from P furiosus in order toimprove the ligation efficiency with the thermostability [103]In Section 44 we showed that the C-terminal helix in theclosed structure observed in PfuLig connects the AdD andthe OBD via five polar or ionic interactions (Figure 13(b)) Incontrast the crystal structures of hLigI and SsoLig revealedthat the relative arrangements of the OBD and the AdDare quite different from that of PfuLig (Figure 9(a)) Takentogether we hypothesized that the binding efficiency ofthe DNA ligase to the DNA substrate would increase byreducing the ionic interactions between the AdD and theOBD resulting in improved ligation efficiency The ionicresidues involved in the interactions between the AdD and

the OBD were selected and mutated to create a series ofmutants in which certain ionic residues are replaced ordeleted First the series of the alanine mutants (1ala 2alaand 3ala) in which the ionic residues at the C-terminal helixare replaced with alanine residues were prepared and theseries of deletion mutants (d4 d8 and d15) were also createdThen the initial reaction rates of thesemutantsweremeasuredat the maximum temperature (60∘C) The initial reactionrates were increased according to the number of replaced ordeleted ionic residues (Figure 14(a)) [104]

Next we found that the initial reaction rate was largelyimproved when the fourth ionic residue (Asp540) from theC-terminus was mutated in addition to the 3ala mutations(D540A3ala)Therefore the single alaninemutant of Asp540(D540A) was created and assessed the effect of the mutationon the ligation efficiency The reaction rate of D540A wasalmost the same as that of D540A3ala [103] suggestingthat the effect of the mutation of Asp540 dominates theimprovement of the alanine mutations of the ionic residues

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

16 Archaea

80

60

40

20

0Initi

al re

actio

n ra

te (p

mol

min

)

WT 1ala 2ala 3ala d4 d8 d5

(a)

Initi

al re

actio

n ra

te (p

mol

min

) 250

150

100

50

0

200

WT

3ala

D54

0A3

ala

D54

0A

D54

0S

D54

0R

D54

0K

(b)

0153045

7590

105120135

60

150

D540R

D540S D540AWT

D540K

Liga

tion

(pm

ol)

15min

20 4030 60 7050 8010 90Temperature (∘C)

(c)

20 4030 60 7050 80

80

0102030

506070

40Li

gatio

n (p

mol

)

10 90

D540R

D540S D540AWT

D540K

120min

Temperature (∘C)

(d)

Figure 14 Nick-joining activities of the wild type and mutant proteins (a) Initial reaction rates for the wild type (WT) K558A (1ala)K554AK558A (2ala) Q547AK554AK558A (3ala) C-terminal 4 residue-deleted mutant d4 (open circles) d8 (open squares) and d15 (opentriangles) enzymes represented by time course data (b) Initial reaction rates forWT 3ala D540A3ala D540A D540S D540R and D540K(c) Nick-joining activities of Asp540 mutants at each temperature for 15min The extents of nick ligation by D540A (open circles) D540S(open squares) D540R (open triangles) andD540K (open diamonds) were plotted as a function of time (d) Nick-joining activities of Asp540mutants at each temperature for 120min Error bars represent the standard deviation (119899 = 3) [103]

on the C-terminal helix Asp540 is located at the AdD-interacting surface of the OBD and forms an ionic pair withArg414 in the vicinity of the AMP binding site in the AdD(Figure 12)

A series of Asp540 mutants in which Asp540 wasreplaced with serine (D540S) lysine (D540K) and arginine(D540R) were also constructed and their ligation efficiencieswere evaluated As a result the initial reaction rates ofD540K and D540R were similarly the fastest among all of themutants followed byD540S thenD540A andfinally thewildtype (Figure 14(b)) The evaluation of the ligation efficienciesof these Asp540 mutants over the broad temperature rangefrom 20 to 80∘C revealed that D540K and D540R werealso the most productive Thus we successfully generatedthe PfuLig mutant with highly improved ligation efficiencyover a broad temperature range while maintaining its usefulthermostability by replacing Asp540 with a basic residuesuch as lysine or arginine (Figures 14(c) and 14(d)) [103]

Further investigation into the effects of the replacement ofAsp540 with a basic residue on the ligation process revealedthat the replacement contributed to both the adenylylationand DNA binding steps These results suggested that theincrease in the number of basic residues in the proximity ofthe active site was advantageous for the adenylylation step inwhich the negatively charged pyrophosphate is pulled awayfrom the ATP molecule in the active site pocket and thatthe alteration to the basic residue was also efficient for theAdDOBDdomain opening caused by the repulsion betweeneither Arg540 or Lys 540 and Arg414 which formed the ionpair with Asp540 (Figure 12) [103]

6 Conclusions

Archaea live in extreme conditions even in the temperaturerange of 80∘C to 100∘C and have yielded many usefulenzymes for gene technology such as hyperthermostable

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 17

DNA polymerases and DNA ligases In this review we havedescribed the utilization of thermostable DNA ligases incommon molecular biology protocols and the structuralmechanism of DNA ligase and shown some examples ofprotein-engineered DNA ligases Improvements of theseenzymes are potentially effective for advancing the existingmethods mentioned above and beneficial for constructingnovel biotechnological methods Several protein engineeringstrategies such as fusion with other proteins or site-directmutagenesis based on structural information may facilitatethe construction of application-specific DNA ligases in thefuture The enzymes can be improved artificially basedon their three-dimensional structures even if they havenaturally evolved for a long period of time

As many Archaea thrive in extreme environments it willbe very interesting to learn how the fidelity mechanisms ofthese extremophilic organisms have adapted to overcomethese harsh conditions Therefore archaeal enzymes willcontinue to play an important role in future research and willbe employed in numerous aspects of gene technology

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor K Morikawa (Kyoto University)and Dr S Ishino (Kyushu University) for their supportYoshizumi Ishino was supported by Grants-in-Aid fromthe Ministry of Education Culture Sports Science andTechnology of Japan (Grant nos 23310152 and 26242075)Theauthors are also grateful to John Wiley amp Sons Ltd for thepermission for the reuse of the figure (Figure 14) from theprevious paper [103]

References

[1] B Weiss and C C Richardson ldquoEnzymatic breakage andjoining of deoxyribonucleic acid I Repair of single-strandbreaks in DNA by an enzyme system from Escherichia coliinfected with T4 bacteriophagerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 57 no4 pp 1021ndash1028 1967

[2] H-M Eun ldquoDNA ligasesrdquo in Enzymology Primer for Recombi-nant DNA Technology pp 109ndash133 Academic Press San DiegoCalif USA 1996

[3] J M Pascal ldquoDNA and RNA ligases structural variations andsharedmechanismsrdquoCurrent Opinion in Structural Biology vol18 no 1 pp 96ndash105 2008

[4] S Shuman and B Schwer ldquoRNA capping enzyme and DNAligase a superfamily of covalent nucleotidyl transferasesrdquoMole-cular Microbiology vol 17 no 3 pp 405ndash410 1995

[5] S Shuman ldquoClosing the gap on DNA ligaserdquo Structure vol 4no 6 pp 653ndash656 1996

[6] U Landegren R Kaiser J Sanders and L Hood ldquoA ligase-med-iated gene detection techniquerdquo Science vol 241 no 4869 pp1077ndash1080 1988

[7] O SoderbergMGullbergM Jarvius et al ldquoDirect observationof individual endogenous protein complexes in situ by proxim-ity ligationrdquo Nature Methods vol 3 no 12 pp 995ndash1000 2006

[8] H S Subramanya A J Doherty S R Ashford and D BWigley ldquoCrystal structure of an ATP-dependent DNA ligasefrom bacteriophage T7rdquo Cell vol 85 no 4 pp 607ndash615 1996

[9] J M Pascal P J OrsquoBrien A E Tomkinson and T Ellen-berger ldquoHumanDNA ligase I completely encircles and partiallyunwinds nicked DNArdquo Nature vol 432 no 7016 pp 473ndash4782004

[10] J M Pascal O V Tsodikov G L Hura et al ldquoA flexible inter-face between DNA ligase and PCNA supports conformationalswitching and efficient ligation of DNArdquoMolecular Cell vol 24no 2 pp 279ndash291 2006

[11] HNishida S Kiyonari Y Ishino andKMorikawa ldquoThe closedstructure of an archaeal DNA ligase from Pyrococcus furiosusrdquoJournal of Molecular Biology vol 360 no 5 pp 956ndash967 2006

[12] S B Zimmerman J W Little C K Oshinsky and M GellertldquoEnzymatic joining of DNA strands a novel reaction of diphos-phopyridine nucleotiderdquoProceedings of theNational Academy ofSciences of the United States of America vol 57 no 6 pp 1841ndash1848 1967

[13] I R Lehman ldquoDNA ligase structure mechanism and func-tionrdquo Science vol 186 no 4166 pp 790ndash797 1974

[14] M J Engler and C C Richardson ldquoDNA ligasesrdquo in TheEnzymes P D Boyer Ed vol 15 pp 3ndash29 Academic Press NewYork NY USA 1982

[15] P Sadowski B Ginsberg A Yudelevich L Feiner and JHurwitz ldquoEnzymatic mechanisms of the repair and breakageof DNArdquo Cold Spring Harbor Symposia on Quantitative Biologyvol 33 pp 165ndash177 1968

[16] T Lindahl and R D Wood ldquoQuality control by DNA repairrdquoScience vol 286 no 5446 pp 1897ndash1905 1999

[17] S Waga and B Stillman ldquoThe DNA replication fork in eukary-otic cellsrdquo Annual Review of Biochemistry vol 67 pp 721ndash7511998

[18] T Ellenberger and A E Tomkinson ldquoEukaryotic DNA ligasesstructural and functional insightsrdquo Annual Review of Biochem-istry vol 77 pp 313ndash338 2008

[19] A Wilkinson J Day and R Bowater ldquoBacterial DNA ligasesrdquoMolecular Microbiology vol 40 no 6 pp 1241ndash1248 2001

[20] V Sriskanda R W Moyer and S Shuman ldquoNAD+-dependentDNA ligase encoded by a eukaryotic virusrdquo The Journal ofBiological Chemistry vol 276 no 39 pp 36100ndash36109 2001

[21] Z A Wood R S Sabatini and S L Hajduk ldquoRNA ligasepicking up the piecesrdquo Molecular Cell vol 13 no 4 pp 455ndash456 2004

[22] L KWang and S Shuman ldquoStructure-function analysis of yeasttRNA ligaserdquo RNA vol 11 no 6 pp 966ndash975 2005

[23] R Sawaya and S Shuman ldquoMutational analysis of the guanylyl-transferase component ofmammalianmRNAcapping enzymerdquoBiochemistry vol 42 no 27 pp 8240ndash8249 2003

[24] S Shuman ldquoDNA ligases progress and prospectsrdquoThe Journalof Biological Chemistry vol 284 no 26 pp 17365ndash17369 2009

[25] V Sriskanda and S Shuman ldquoChlorella virus DNA ligase nickrecognition and mutational analysisrdquo Nucleic Acids Researchvol 26 no 2 pp 525ndash531 1998

[26] V Sriskanda and S Shuman ldquoRole of nucleotidyltransferasemotifs I III and IV in the catalysis of phosphodiester bond for-mation by Chlorella virus DNA ligaserdquo Nucleic Acids Researchvol 30 no 4 pp 903ndash911 2002

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

18 Archaea

[27] V Sriskanda and S Shuman ldquoMutational analysis of Chlorellavirus DNA ligase Catalytic roles of domain I and motif VIrdquoNucleic Acids Research vol 26 no 20 pp 4618ndash4625 1998

[28] P Samai and S Shuman ldquoKinetic analysis of DNA strand join-ing by Chlorella virus DNA ligase and the role of nucleotidyl-transferase motif VI in ligase adenylylationrdquo The Journal ofBiological Chemistry vol 287 no 34 pp 28609ndash28618 2012

[29] C A Foy and H C Parkes ldquoEmerging homogeneous DNA-based technologies in the clinical laboratoryrdquo Clinical Chem-istry vol 47 no 6 pp 990ndash1000 2001

[30] R C Conaway and I R Lehman ldquoADNAprimase activity asso-ciated with DNA polymerase alpha from Drosophila melano-gaster embryosrdquoProceedings of theNational Academy of Sciencesof theUnited States of America vol 79 no 8 pp 2523ndash2527 1982

[31] H A Erlich ldquoPolymerase chain reactionrdquo Journal of ClinicalImmunology vol 9 no 6 pp 437ndash447 1989

[32] D S LevinW Bai N YaoM OrsquoDonnell and A E TomkinsonldquoAn interaction between DNA ligase I and proliferating cellnuclear antigen implications for Okazaki fragment synthesisand joiningrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 94 no 24 pp 12863ndash128681997

[33] N G Copeland N A Jenkins and D L Court ldquoRecombineer-ing a powerful new tool for mouse functional genomicsrdquoNature Reviews Genetics vol 2 no 10 pp 769ndash779 2001

[34] D Y Wu and R B Wallace ldquoSpecificity of the nick-closingactivity of bacteriophage T4 DNA ligaserdquo Gene vol 76 no 2pp 245ndash254 1989

[35] AMAlves and F J Carr ldquoDot blot detection of pointmutationswith adjacently hybridising synthetic oligonucleotide probesrdquoNucleic Acids Research vol 16 no 17 article 8723 1988

[36] D A Nickerson R Kaiser S Lappin J Stewart L Hood andU Landegren ldquoAutomated DNA diagnostics using an ELISA-based oligonucleotide ligation assayrdquoProceedings of theNationalAcademy of Sciences of the United States of America vol 87 no22 pp 8923ndash8927 1990

[37] I A Beck M Mahalanabis G Pepper et al ldquoRapid and sensi-tive oligonucleotide ligation assay for detection of mutationsin human immunodeficiency virus type 1 associated withhigh-level resistance to protease inhibitorsrdquo Journal of ClinicalMicrobiology vol 40 no 4 pp 1413ndash1419 2002

[38] F Barany ldquoThe ligase chain reaction in a PCR worldrdquo GenomeResearch vol 1 no 1 pp 5ndash16 1991

[39] F Barany ldquoGenetic disease detection and DNA amplificationusing cloned thermostable ligaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 88 no1 pp 189ndash193 1991

[40] M Khanna W Cao M Zirvi P Paty and F Barany ldquoLigasedetection reaction for identification of low abundance muta-tionsrdquo Clinical Biochemistry vol 32 no 4 pp 287ndash290 1999

[41] H H Lee ldquoLigase chain reactionrdquo Biologicals vol 24 no 3 pp197ndash199 1996

[42] D Y Wu and R B Wallace ldquoThe ligation amplificationreaction (LAR)-amplification of specific DNA sequences usingsequential rounds of template-dependent ligationrdquo Genomicsvol 4 no 4 pp 560ndash569 1989

[43] S Minamitani S Nishiguchi T Kuroki S Otani and TMonna ldquoDetection by ligase chain reaction of precore mutantof hepatitis B virusrdquoHepatology vol 25 no 1 pp 216ndash222 1997

[44] V D Karthigesu M Mendy M Fortuin H C Whittle C RHoward and LM C Allison ldquoThe ligase chain reaction distin-guishes hepatitis B virus S-gene variantsrdquo FEMS MicrobiologyLetters vol 131 no 2 pp 127ndash132 1995

[45] C Osiowy ldquoSensitive detection of HBsAG mutants by a gapligase chain reaction assayrdquo Journal of ClinicalMicrobiology vol40 no 7 pp 2566ndash2571 2002

[46] M Zirvi T Nakayama G Newman T McCaffrey P Paty andF Barany ldquoLigase-based detection of mononucleotide repeatsequencesrdquo Nucleic Acids Research vol 27 no 24 pp e40indashe40viii 1999

[47] C A Batt PWagnerMWiedmann and R Gilbert ldquoDetectionof bovine leukocyte adhesion deficiency by nonisotopic ligasechain reactionrdquo Animal Genetics vol 25 no 2 pp 95ndash98 1994

[48] K Abravaya J J Carrino S Muldoon and H H Lee ldquoDetec-tion of point mutations with a modified ligase chain reaction(Gap-LCR)rdquo Nucleic Acids Research vol 23 no 4 pp 675ndash6821995

[49] V L Wilson Q Wei K R Wade et al ldquoNeedle-in-a-haystackdetection and identification of base substitution mutations inhuman tissuesrdquo Mutation Research vol 406 no 2ndash4 pp 79ndash100 1999

[50] C Niederhauser L Kaempf and I Heinzer ldquoUse of the ligasedetection reaction-polymerase chain reaction to identify pointmutations in extended-spectrum beta-lactamasesrdquo EuropeanJournal of Clinical Microbiology and Infectious Diseases vol 19no 6 pp 477ndash480 2000

[51] C Bourgeois N Sixt J B Bour and P Pothier ldquoValue of a ligasechain reaction assay for detection of ganciclovir resistance-related mutation 594 in UL97 gene of human cytomegalovirusrdquoJournal of Virological Methods vol 67 no 2 pp 167ndash175 1997

[52] M Szemes P Bonants M de Weerdt J Baner U Landegrenand C D Schoen ldquoDiagnostic application of padlock probes-multiplex detection of plant pathogens using universalmicroar-raysrdquo Nucleic Acids Research vol 33 no 8 article e70 2005

[53] M Nilsson H Malmgren M Samiotaki M Kwiatkowski B PChowdhary and U Landegren ldquoPadlock probes circularizingoligonucleotides for localized DNA detectionrdquo Science vol 265no 5181 pp 2085ndash2088 1994

[54] M Nilsson K Krejci J Koch M Kwiatkowski P GustavssonandU Landegren ldquoPadlock probes reveal single-nucleotide dif-ferences parent of origin and in situ distribution of centromericsequences in human chromosomes 13 and 21rdquo Nature Geneticsvol 16 no 3 pp 252ndash255 1997

[55] D-O Antson M Mendel-Hartvig U Landegren and MNilsson ldquoPCR-generated padlock probes distinguish homolo-gous chromosomes through quantitative fluorescence analysisrdquoEuropean Journal of Human Genetics vol 11 no 5 pp 357ndash3632003

[56] A Fire and S-Q Xu ldquoRolling replication of short DNA circlesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 10 pp 4641ndash4645 1995

[57] D Liu S L DaubendiekMA Zillman K Ryan and E T KoolldquoRolling circle DNA synthesis small circular oligonucleotidesas efficient templates for DNA polymerasesrdquo Journal of theAmerican Chemical Society vol 118 no 7 pp 1587ndash1594 1996

[58] S L Daubendiek and E T Kool ldquoGeneration of catalytic RNAsby rolling transcription of synthetic DNA nanocirclesrdquo NatureBiotechnology vol 15 no 3 pp 273ndash277 1997

[59] P M Lizardi X Huang Z Zhu P Bray-Ward D C Thomasand D C Ward ldquoMutation detection and single-molecule

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Archaea 19

counting using isothermal rolling-circle amplificationrdquo NatureGenetics vol 19 no 3 pp 225ndash232 1998

[60] J Baner M Nilsson M Mendel-Hartvig and U LandegrenldquoSignal amplification of padlock probes by rolling circle repli-cationrdquo Nucleic Acids Research vol 26 no 22 pp 5073ndash50781998

[61] C Larsson J Koch A Nygren et al ldquoIn situ genotyping indi-vidual DNA molecules by target-primed rolling-circle amplifi-cation of padlock probesrdquoNatureMethods vol 1 no 3 pp 227ndash232 2004

[62] X-B Zhong P M Lizardi X-H Huang P L Bray-Ward andD C Ward ldquoVisualization of oligonucleotide probes and pointmutations in interphase nuclei and DNA fibers using rollingcircle DNA amplificationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 98 no 7 pp3940ndash3945 2001

[63] O Soderberg K-J Leuchowius M Gullberg et al ldquoCharacter-izing proteins and their interactions in cells and tissues usingthe in situ proximity ligation assayrdquoMethods vol 45 no 3 pp227ndash232 2008

[64] M Margulies M Egholm W E Altman et al ldquoGenomesequencing in microfabricated high-density picolitre reactorsrdquoNature vol 437 no 7057 pp 376ndash380 2005

[65] D R Bentley ldquoWhole-genome re-sequencingrdquoCurrent Opinionin Genetics amp Development vol 16 no 6 pp 545ndash552 2006

[66] E R Mardis ldquoNext-generation DNA sequencing methodsrdquoAnnual Review of Genomics and Human Genetics vol 9 pp387ndash402 2008

[67] M Ronaghi M Uhlen and P Nyren ldquoA sequencing methodbased on real-time pyrophosphaterdquo Science vol 281 no 5375pp 363ndash365 1998

[68] OMorozova andMAMarra ldquoApplications of next-generationsequencing technologies in functional genomicsrdquo Genomicsvol 92 no 5 pp 255ndash264 2008

[69] J Shendure G J Porreca N B Reppas et al ldquoAccurate multi-plex colony sequencing of an evolved bacterial genomerdquo Sci-ence vol 309 no 5741 pp 1728ndash1732 2005

[70] J Shendure and H Ji ldquoNext-generation DNA sequencingrdquoNature Biotechnology vol 26 no 10 pp 1135ndash1145 2008

[71] J J Dunn and F W Studier ldquoNucleotide sequence from thegenetic left end of bacteriophage T7 DNA to the beginning ofgene 4rdquo Journal of Molecular Biology vol 148 no 4 pp 303ndash330 1981

[72] D E Barnes L H Johnston K I Kodama A E Tomkinson DD Lasko and T Lindahl ldquoHuman DNA ligase I cDNA clon-ing and functional expression in Saccharomyces cerevisiaerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 87 no 17 pp 6679ndash6683 1990

[73] A Kletzin ldquoMolecular characterisation of a DNA ligase geneof the extremely thermophilic archaeonDesulfurolobus ambiva-lens shows close phylogenetic relationship to eukaryotic ligasesrdquoNucleic Acids Research vol 20 no 20 pp 5389ndash5396 1992

[74] A J Doherty S R AshfordH S Subramanya andD BWigleyldquoBacteriophage T7 DNA ligase overexpression purificationcrystallization and characterizationrdquo The Journal of BiologicalChemistry vol 271 no 19 pp 11083ndash11089 1996

[75] M Odell V Sriskanda S Shuman and D B Nikolov ldquoCrystalstructure of eukaryotic DNA ligase-adenylate illuminates themechanism of nick sensing and strand joiningrdquoMolecular Cellvol 6 no 5 pp 1183ndash1193 2000

[76] A J Doherty and T R Dafforn ldquoNick recognition by DNAligasesrdquo Journal of Molecular Biology vol 296 no 1 pp 43ndash562000

[77] V Sriskanda and S Shuman ldquoRole of nucleotidyl transferasemotif V in strand joining by Chlorella virus DNA ligaserdquo TheJournal of Biological Chemistry vol 277 no 12 pp 9661ndash96672002

[78] D Suck ldquoCommon fold common function common originrdquoNature Structural ampMolecular Biology vol 4 no 3 pp 161ndash1651997

[79] A G Murzin ldquoOB(oligonucleotideoligosaccharide binding)-fold common structural and functional solution for non-homologous sequencesrdquo The EMBO Journal vol 12 no 3 pp861ndash867 1993

[80] K Hakansson A J Doherty S Shuman and D B WigleyldquoX-ray crystallography reveals a large conformational changeduring guanyl transfer by mRNA capping enzymesrdquo Cell vol89 no 4 pp 545ndash553 1997

[81] A V Cherepanov and S de Vries ldquoDynamicmechanism of nickrecognition by DNA ligaserdquo European Journal of Biochemistryvol 269 no 24 pp 5993ndash5999 2002

[82] D J Kim O Kim H-W Kim H S Kim S J Lee and S WSuh ldquoATP-dependent DNA ligase from Archaeoglobus fulgidusdisplays a tightly closed conformationrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 65 no 6 pp 544ndash550 2009

[83] T Petrova E Y Bezsudnova K M Boyko et al ldquoATP-depend-ent DNA ligase from Thermococcus sp 1519 displays a newarrangement of the OB-fold domainrdquo Acta CrystallographicaSection F Structural Biology and Crystallization Communica-tions vol 68 no 12 pp 1440ndash1447 2012

[84] M C Cardoso C Joseph H P Rahn R Reusch B Nadal-Ginard andH Leonhardt ldquoMapping and use of a sequence thattargets DNA ligase I to sites of DNA replication in vivordquo TheJournal of Cell Biology vol 139 no 3 pp 579ndash587 1997

[85] C Prigent D D Lasko K Kodama J R Woodgett andT Lindahl ldquoActivation of mammalian DNA ligase I throughphosphorylation by casein kinase IIrdquo The EMBO Journal vol11 no 8 pp 2925ndash2933 1992

[86] N Keppetipola and S Shuman ldquoCharacterization of a ther-mophilic ATP-dependent DNA ligase from the euryarchaeonPyrococcus horikoshiirdquo Journal of Bacteriology vol 187 no 20pp 6902ndash6908 2005

[87] H Echols and M F Goodman ldquoFidelity mechanisms in DNAreplicationrdquoAnnual Review of Biochemistry vol 60 pp 477ndash5111991

[88] YWang D E Prosen L Mei J C SullivanM Finney and P BVander Horn ldquoA novel strategy to engineer DNA polymerasesfor enhanced processivity and improved performance in vitrordquoNucleic Acids Research vol 32 no 3 pp 1197ndash1207 2004

[89] T Kuroita H Matsumura N Yokota et al ldquoStructural mech-anism for coordination of proofreading and polymerase activi-ties in archaealDNApolymerasesrdquo Journal ofMolecular Biologyvol 351 no 2 pp 291ndash298 2005

[90] B H Pheiffer and S B Zimmerman ldquoPolymer-stimulatedligation enhanced blunt- or cohesive-end ligation of DNAor deoxyribooligonudcleotides by T4 DNA ugase in polymersolutionsrdquo Nucleic Acids Research vol 11 no 22 pp 7853ndash78711983

[91] V Sgaramella J H van de Sande and H G Khorana ldquoStudieson polynucleotides C A novel joining reaction catalyzed by the

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

20 Archaea

T4-polynucleotide ligaserdquo Proceedings of the National Academyof Sciences of theUnited States of America vol 67 no 3 pp 1468ndash1475 1970

[92] R H Wilson S K Morton H Deiderick et al ldquoEngineeredDNA ligases with improved activities in vitrordquo Protein Engineer-ing Design amp Selection vol 26 no 7 pp 471ndash478 2013

[93] A Sugino H M Goodman H L Heyneker J Shine H WBoyer and N R Cozzarelli ldquoInteraction of bacteriophage T4RNA and DNA ligases in joining of duplex DNA at base pairedendsrdquo The Journal of Biological Chemistry vol 252 no 11 pp3987ndash3994 1977

[94] G J S Lohman S Tabor and N M Nichols ldquoDNA ligasesrdquo inCurrent Protocols in Molecular Biology vol 94 chapter 3 unit314 pp 1ndash7 2011

[95] K Harada and L E Orgel ldquoUnexpected substrate specificityof T4 DNA ligase revealed by in vitro selectionrdquo Nucleic AcidsResearch vol 21 no 10 pp 2287ndash2291 1993

[96] C Goffin V Bailly and W G Verly ldquoNicks 31015840 or 51015840 to AP sitesor to mispaired bases and one-nucleotide gaps can be sealedby T4 DNA ligaserdquo Nucleic Acids Research vol 15 no 21 pp8755ndash8771 1987

[97] J Luo D E Bergstrom and F Barany ldquoImproving the fidelity ofThermus thermophilusDNA ligaserdquo Nucleic Acids Research vol24 no 15 pp 3071ndash3078 1996

[98] W A Beard S J Stahl H-R Kim et al ldquoStructurefunctionstudies of human immunodeficiency virus type 1 reverse tran-scriptase Alanine scanning mutagenesis of an 120572-helix in thethumb subdomainrdquo The Journal of Biological Chemistry vol269 no 45 pp 28091ndash28097 1994

[99] L J Reha-Krantz R L Nonay and S Stocki ldquoBacteriophage T4DNA polymerase mutations that confer sensitivity to the PPianalog phosphonoacetic acidrdquo Journal of Virology vol 67 no 1pp 60ndash66 1993

[100] L J Reha-Krantz and R L Nonay ldquoMotif A of bacteriophageT4 DNA polymerase role in primer extension and DNAreplication fidelityrdquoThe Journal of Biological Chemistry vol 269no 8 pp 5635ndash5643 1994

[101] Q Dong W C Copeland and T S-F Wang ldquoMutationalstudies of human DNA polymeraserdquo The Journal of BiologicalChemistry vol 268 no 32 pp 24163ndash24174 1993

[102] W C Copeland N K Lam and T S-F Wang ldquoFidelitystudies of the human DNA polymerase 120572 The most conservedregion among120572-likeDNApolymerases is responsible formetal-induced infidelity in DNA synthesisrdquo The Journal of BiologicalChemistry vol 268 no 15 pp 11041ndash11049 1993

[103] M Tanabe S Ishino M Yohda K Morikawa Y Ishino andH Nishida ldquoStructure-based mutational study of an archaealDNA ligase towards improvement of ligation activityrdquo Chem-BioChem vol 13 no 17 pp 2575ndash2582 2012

[104] M Tanabe S Ishino Y Ishino and H Nishida ldquoMutationsof Asp540 and the domain-connecting residues synergisticallyenhance Pyrococcus furiosus DNA ligase activityrdquo FEBS Lettersvol 588 no 2 pp 230ndash235 2014

[105] E F Pettersen T D Goddard C C Huang et al ldquoUCSFChimeramdasha visualization system for exploratory research andanalysisrdquo Journal of Computational Chemistry vol 25 no 13pp 1605ndash1612 2004

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology