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Molecular Biology ReportsAn International Journal on Molecularand Cellular Biology ISSN 0301-4851 Mol Biol RepDOI 10.1007/s11033-016-3949-3

Engineering TaqII bifunctionalendonuclease DNA recognition fidelity: theeffect of a single amino acid substitutionwithin the methyltransferase catalytic site

Agnieszka Zylicz-Stachula, JoannaZebrowska, Edyta Czajkowska,Weronika Wrese, Ewa Sulecka & PiotrM. Skowron

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ORIGINAL ARTICLE

Engineering TaqII bifunctional endonuclease DNA recognitionfidelity: the effect of a single amino acid substitutionwithin the methyltransferase catalytic site

Agnieszka Zylicz-Stachula1 • Joanna Zebrowska1 • Edyta Czajkowska1 •

Weronika Wrese1 • Ewa Sulecka1 • Piotr M. Skowron1

Received: 14 July 2015 / Accepted: 8 February 2016

� Springer Science+Business Media Dordrecht 2016

Abstract The aim of this study was to improve a useful

molecular tool—TaqII restriction endonuclease-methyl-

transferase—by rational protein engineering, as well as to

show an application of our novel method of restriction

endonuclease activity modulation through a single amino

acid change in the NPPY motif of methyltransferase. An

amino acid change was introduced using site-directed

mutagenesis into the taqIIRM gene. The mutated gene was

expressed in Escherichia coli. The protein variant was

purified and characterized. Previously, we described a

TspGWI variant with an amino acid change in the

methyltransferase motif IV. Here, we investigate a com-

plex, pleiotropic effect of an analogous amino acid change

on its homologue—TaqII. The methyltransferase activity is

reduced, but not abolished, while TaqII restriction

endonuclease can be reactivated by sinefungin, with an

increased DNA recognition fidelity. The general method

for engineering of the IIS/IIC/IIG restriction endonuclease

activity/fidelity is developed along with the generation of

an improved TaqII enzyme for biotechnological applica-

tions. A successful application of our novel strategy for

restriction endonuclease activity/fidelity alteration, based

on bioinformatics analyses, mutagenesis and the use of

cofactor-analogue activity modulation, is presented.

Keywords Restriction endonuclease �Methyltransferase � Type IIS � Type IIG �Gene mutagenesis

Introduction

Restriction endonucleases (REases) bring interest both as

extensively used molecular tools and models for protein-

DNA interaction studies. Thermostable REases are robust

enzymes due to their high thermal stability and long shelf-

life as commercial products [1]. They are indispensable in

procedures based on the PCR technique, requiring high

catalytic activity at elevated temperatures, exemplified by:

the polymerase-endonuclease amplification reaction

(PEAR) [2], Thermostable restriction enzyme PCR

screening [3] or restriction endonuclease-mediated real-

time PCR digestion (RTD-PCR) [4].

Due to extensive bioinformatic studies, an increasing

number of atypical restriction-modification (RM) systems

are being discovered, including multifunctional enzymes,

exemplified by REases-regulatory C protein fusions [5] or

the Thermus sp. family of Type IIS/IIG/IIC restriction

endonucleases/methyltransferases (REases–MTases)

fusions [6, 7].

The Thermus sp. family groups atypical enzymes which

also exhibit some of the features characteristic for Types I

& Agnieszka Zylicz-Stachula

a.zylicz-stachula@ug.edu.pl

Joanna Zebrowska

asia.zebrowska87@gmail.com

Edyta Czajkowska

edyta_czajkowska@wp.pl

Weronika Wrese

werusia22@wp.pl

Ewa Sulecka

ewa.sulecka@ug.edu.pl

Piotr M. Skowron

piotr.skowron@ug.edu.pl

1 Department of Molecular Biotechnology, Institute for

Environmental and Human Health Protection, Division of

Chemistry, University of Gdansk, Wita Stwosza 63,

80-308 Gdansk, Poland

123

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DOI 10.1007/s11033-016-3949-3

Author's personal copy

and III REases [8]. The family includes bifunctional

REases–MTases of nearly identical, atypical molecular

sizes (approx. 120 kDa), substantial similarities in recog-

nition sequences, identity of cleavage sites, partial amino

acid (aa) sequence similarity (unusual for Type II REases),

fused protein domain organizations, quaternary structure

(monomers) and cleavage stimulation by S-adenosylme-

thionine (SAM) or its structural analogue sinefungin (SIN)

[7, 9, 10].

Despite biochemical similarity, significant differences

between the aa compositions of the proteins belonging to

the Thermus sp. family were revealed, which resulted in

defining two related subfamilies [11]: (1) the TspGWI

subfamily, including RM.TaqII [7, 10, 12–14],

RM.TspGWI [6, 9, 15] and (2) the TspDTI subfamily,

including RM.TspDTI [11], RM.TsoI [11, 16], the iso-

schizomer pair RM.TthHB27I/Tth111II [17–19]. Several

homologous proteins from mesophilic bacteria, present in

databases (REBASE: http://rebase.neb.com), apparently

belong to the Thermus family of related enzymes [7, 16].

Many more putative, homologous genes with uncharac-

terized coded proteins are listed in REBASE for both

TspGWI and TspDTI subfamilies (http://rebase.neb.com).

Interestingly, the enzymes from the Thermus sp. family

display significant differences in their response to SAM

and its analogues, among others. Remarkably, the response

of a REase to SAM/SIN/S-adenosylhomocysteine (SAH)

can be modulated through aa substitutions in the catalytic

centre of a fused MTase [9, 20].

The prototype TaqII REase from the TspGWI subfamily is

strongly affected by SAM or its analogue SIN [10]. We

reported previously an unprecedented phenomenon—a SIN-

mediated TaqII REase specificity change (‘affinity star’

activity) from the 6 bp recognition site 50-GACCGA-30 [11/

9] to a combined 2.9 bp recognition site [10]. Since the

specificity change was caused by a cofactor analogue, we

designated this phenomenon ‘affinity star’ specificity, in

contrast to the classic ‘star’ activity of REases, caused by

altered standard chemical reaction conditions (pH, salt,

divalent cation composition and enzyme concentration).

Based on this chemically induced relaxation of TaqII and

TspGWI DNA recognition sequence specificities, we devel-

oped a technology of ultra-frequent DNA fragmentation for

the purpose of representative, genomic DNA libraries [10].

This technology was used to develop a new genomic tool for

the generation of quasi-random DNA libraries [10].

RM.TaqII enzyme exhibits aa sequence similarity to

thermostable RM.TspGWI (64 % identity, 75 % positives,

E value 0.0) and thermolabile RM.RpaI (39 % identity,

53 % positives, E value 0.0) [9,15, REBASE: http://rebase.

neb.com]—one of the highest similarities, reported so far

in the literature for Type II REases, recognizing different

DNA sequences. Another homologous gene, annotated as

taqIIRM, was found in Anabaena sp. 90 genomic DNA

(45 % identity, 62 % positives, E value 0.0) [21]. Cur-

rently, the TspGWI subfamily comprises of over 63 dif-

ferent putative members, which provides a

suitable platform for comparative DNA-recognition studies

and analysis of the protein thermostability determi-

nants (REBASE: http://rebase.neb.com).

Bioinformatics analysis of the homologous genes cod-

ing for REases from the TspGWI-like subfamily coupled

with the experimental confirmation by site-directed muta-

genesis and biochemical approach allowed for the deter-

mination of distinct functional domains [9, 15] and

construction of a series of mutants in the tspGWIRM gene

[9, 20] and its homologue taqIIRM gene (not shown).

Whilst investigating the RM.TspGWI variants in more

detail, we found that a single aa substitution of N to A at

position 473 in motif IV of the MTase domain not only

eliminated MTase activity but also decreased the remain-

ing REase activity to less than 0.8 % compared to an

unaltered, recombinant wt RM.TspGWI protein, indicating

the existence of long distance interdomain communication

[20] (Table 1). This effect could be supressed by the

addition of SIN to the reaction mixture, resulting in an

increase in the TspGWI N473A REase activity to over

25–50 % in comparison to the wt protein, essentially

restoring its functionality (Table 1). As SIN is not a methyl

group donor and thus remains unchanged by both the wt

and mutant enzyme, we hypothesized that it acts as an

allosteric effector bringing polypeptide chains to stable and

properly spaced positions, needed to maintain adequate

tertiary structure for remote DNA scission catalysis [20].

Our findings are in agreement with the results of Sarrade-

Loucheur et al. [22], who showed that bifunctional RM.

BpuSI—an enzyme with a domain organization resembling

the one of enzymes from the Thermus family—underwent

substantial conformational changes in the presence of SIN,

DNA substrate and Ca2? [22]. After SIN binding, the

enzyme adopted a unique conformation which promoted

cleavage, probably due to the increased binding affinity to

the specific DNA substrate as proposed by the authors [22].

Further, there have been a number of studies on how SAM

or related compounds affect MTase structure [23]. These

studies showed that SAM and DNA binding are not

entirely independent. This would presumably also apply to

the REase–MTase fusion proteins.

This study on the TaqII N472A protein variant is a

continuation of our previous studies on the TspGWI

enzyme [20]. It concerns a novel method of DNA recog-

nition fidelity modification through a single aa substitution

in the catalytic centre of an MTase (motif IV of MTase-

NPPY). In this paper, we develop a method for rational

protein engineering of bifunctional REases–MTases for

DNA biotechnology applications.

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Materials and methods

Bacterial strains, plasmids, media and reagents

A wild-type (wt), recombinant TaqII protein expression

plasmid (pRZ-wt-taqIIRM) was previously constructed

[14]. Marathon DNA Polymerase was from A&A

Biotechnology (Gdansk, Poland). PCR primer synthesis

was performed at Genomed (Warsaw, Poland). DNA,

protein markers, PierceTM BCA Protein Assay Kit and the

Miniprep DNA Purification Kit were from Thermo Scien-

tific (Fermentas, Vilnius, Lithuania). Chromatographic ion

exchange and affinity columns were from GE Medical

Systems Polska Ltd. (Warsaw, Poland). All other reagents

were purchased from Sigma-Aldrich (St Louis, MO, USA).

Escherichia coli (E. coli) Top10 {F-mcrA D(mrr-hsdRMS-

mcrBC) /80lacZDM15 DlacX74 nupG recA1 araD139

D(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 k-}

(Invitrogen, Carlsbad, CA, USA) was used for the selection

of clones after mutagenesis. E. coli BL21(DE3) {F-ompT

gal dcm lon hsdSB(rB� ,mB� ) k (DE3)} (Novagen, Madison,

WI, USA) was employed for taqIIRM and taqIIRM-N472A

gene expression. The predicted DNA cleavage patterns

were prepared using either a trial version of SnapGene

software or the REBsites software.

Site-specific mutagenesis of taqIIRM gene

Site-directed mutagenesis within the recombinant wt

taqIIRM gene was carried out by PCR amplification of the

entire plasmid, with modifications introduced using the

following primers: forward primer 50-TGGTCCTGGGCG

Table 1 Effects of N472/473A substitution in NPPY motif on the activity of homologous TaqII and TspGWI enzymes

Enzyme Recombinant RM.TaqII Recombinant RM.TspGWI

Variant wt N472A wt N473A

MTase (This work; Fig. 3) [20]

Active Eightfold

drop in

activity

Active Lack of activity

REase In buffer A (Fig. 4; this work;

[13]) established for

minimum TaqII REase ‘star’

activity

In the optimal reaction buffer [20]

Without

cofactor

Trace activity Trace

activity

Active Lack of activity

With

SAM

32-fold

allosteric

activation**

Trace

activity

Fourfold drop in activity (in

comparison to the optimal reaction

buffer without SAM or SIN)

Lack of activity

With SIN 64-fold

allosteric

activation***

Trace

activity

Twofold increase in activity (in

comparison to the optimal reaction

buffer without SAM or SIN)

Two to fourfold allosteric activation (only partial

activation of REase: 25–50 % of wt recombinant

TspGWI without cofactor or its analogue)

In buffer B**** (Fig. 5; this

work) for maximum

TaqII REase activity

Without

cofactor

Eightfold

activation**

Fourfold

activation*

With

SAM

16-fold

allosteric

activation**

Eightfold

allosteric

activation*

With SIN 128-fold

allosteric

activation***

32-fold

allosteric

activation*

The studies were performed using the previously described 390 bp DNA fragment [13, 20, 30]. The results presented in this table are not

precisely comparable as RM.TspGWI has different cleavage requirements and is not able to cleave DNA substrate with a single DNA recognition

sequence. The influence of the reaction buffer on N473A TspGWI variant was not investigated

* Barely detectable TaqII REase ‘affinity star’ activity

** Clearly detectable TaqII REase ‘affinity star’ activity

*** Strong TaqII REase ‘affinity star’ activity

**** Trace TaqII REase activity in buffer A without SAM or SIN (Fig. 4a) was used as a reference point for the following calculations

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CGCCCCCCTACGATCGGGTAGAAG-30 and reverse

primer 50-CTTCTACCCGATCGTAGGGGGGCGCCCC

CAGGACCA-30. PCR reactions were performed as

described previously [9]. The resulting recombinant plas-

mids pRZ-taqIIRM-N472A obtained from mutant clones

were verified by DNA sequencing.

Expression of the taqIIRM (N472A) gene under PR

promoter control in E. coli and purification

of the TaqII N472A enzyme

Procedures of recombinant wt RM.TaqII clone culturing,

induction and protein purification were previously described

[14] and applied with modifications to accommodate the

changed properties of the N472A RM.TaqII variant (NPPY

motif IV to APPY). Expression of the mutated gene was

performed in E.coli BL21(DE3) [pRZ-taqIIRM-N472A] in

TB medium [24] supplemented with chloramphenicol

(40 lg/ml) and maltose (0.5 %) at 30 �C with vigorous

aeration. PR promoter induction was obtained through a

rapid temperature shift to 42 �C. The culture was grown at

30 �C until OD600 0.9 was reached, and a temperature shift

was conducted by the addition of TB medium [24], pre-

warmed to 65 �C. A control bacterial culture without

induction of the taqIIRM-N472A gene expression was per-

formed at 30 �C. Moreover, two corresponding control

bacterial cultures were performed using E.coli BL21(DE3)

[pRZ-wt-taqIIRM]. Samples for microscopic analysis were

taken from both E.coli BL21(DE3) [pRZ-wt-taqIIRM] and

[pRZ-taqIIRM-N472A] cultures before induction, 1.5 and 3 h

after induction. Bacteria were immediately stained following

the methylene blue positive staining protocol [24]. Slide

preparations were examined and photographed as previously

described [20]. In case of both non-induced controls, bac-

terial growth was continued for 21 h at 30 �C. The induced

cultures, producing either wt RM.TaqII or TaqII N472A

protein were stopped after 2.5 h when the OD600 reached

about 1.9 and the bacteria were immediately centrifuged.

The centrifugation was performed without pre-cooling to

prevent lysis of the fragile recombinant E. coli cells [pRZ-

taqIIRM-N472A]. Bacterial pellets from non-induced con-

trols and induced cultures were subjected to SDS-PAGE and

the presence of the TaqII protein variants was determined.

The protein variants were purified using the following

purification stages:

(i) Lysis and heat treatment 10 g of bacterial cells

was suspended in 100 ml of buffer A [50 mM

Tris–HCl (pH 7.9 at 25 �C), 0.1 mM EDTA,

50 mM NaCl, 5 % glycerol, 0.5 % Triton-X-100,

5 mM 2-mercaptoethanol (bMe), 1 mM PMSF,

0.5 mg/ml chicken egg lysozyme]. After 30 min

incubation at 4 �C, the lysate was sonicated and

centrifuged. The supernatant was incubated for

30 min at 65 �C. The denatured thermolabile

E. coli proteins were removed by centrifugation.

(ii) Q Sepharose FF chromatography Anion exchange

was conducted in buffer B [20 mM Tris-HCl

(pH 7.0 at 4 �C), 0.1 mM EDTA, 0.25 MgCl2,

100 mM NaCl]. The column was flushed with

buffer B. The proteins bound on the column were

eluted over a 30 CV linear salt gradient from 100

to 500 mM sodium chloride in buffer B. All runs

were carried out at 4 �C. Pooled column fractions

containing the TaqII variants were dialysed

against buffer C [20 mM Tris-HCl (pH 8.0 at

4 �C), 10 mM MgCl2, 80 mM NaCl, 5 % glyc-

erol, 5 mM bMe].

(iii) Heparin agarose chromatography The separation

was conducted using buffer C with included

increasing NaCl concentration steps (mM): 100,

150, 300, 500. TaqII was eluted at 150-300 mM

NaCl. Column fractions containing the enzyme

were dialysed against buffer D [20 mM Tris-HCl

(pH 8.0 at 4 �C), 100 mM NaCl, 0.1 mM EDTA,

0.25 mM MgCl2].

(iv) Size exclusion chromatography The procedure

took advantage of the high molecular weight of

TaqII REase as compared to other E. coli proteins.

A HiLoad 16/600 Superdex 200 prep grade

column (GE Healthcare) was equilibrated in

buffer D and concentrated TaqII preparation was

subjected to molecular sieving. Purified prepara-

tion was dialysed against storage buffer S [20 mM

Tris-HCl (pH 8.0 at 4 �C), 200 mM NaCl; 1 mM

DTT, 50 % glycerol] and stored at -20 �C.

The purity of both enzyme preparations was estimated on a

10 % SDS-PAGE gel. Protein concentrations were deter-

mined using two methods: PierceTM BCA Protein Assay

Kit and Coomassie Brillant Blue stained protein band

densitometric analysis, with the use of a BSA serial dilu-

tion calibration curve (the concentration values were

obtained from a linear standard reflective scan mode with

background correction in UN-SCAN IT GEL for Windows

6.1 data software [v. 6.1, Gel Analysing and Graph Digi-

tizing Software (Silk Scientific Corporation, Orem, UT,

USA)].

MTase activity in a DNA protection assay

The MTase activity of TaqII protein variants was investi-

gated through a DNA protection assay. For that purpose, a

custom PCR substrate containing one RM.TaqII recogni-

tion sequence 50-GACCGA-30 (?) [13] was incubated in

the presence of 200 lM SAM in reaction buffer M: 10 mM

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Tris-HCl pH 7.2 at 70 �C, 10 mM CaCl2, 1 mM DTT. The

reaction volume of 100 ll contained 300 ng of the custom

PCR substrate. The experiments utilized recombinant wt

RM.TaqII and the N472A RM.TaqII variant protein. Titra-

tion experiments were carried out as a series of twofold

dilutions, starting from 1250 ng (10 pmol) down to 78 ng

(0.31 pmol) of both protein variants. After 1 h of incuba-

tion at 70 �C, the reactions were immediately stopped by

the addition of EDTA, SDS and proteinase K. The proteins

were digested for 1 h at 55 �C, phenol–chloroform

extracted and DNA was ethanol-precipitated. DNA was re-

dissolved in the following buffer: 10 mM Tris-HCl pH 7.2

at 70 �C, 10 mM MgCl2, 1 mM DTT, BSA 100 lg/ml in

the presence of 20 lM SAM and challenged with an excess

of wt TaqII REase for 1 h at 70 �C, then the reactions were

immediately stopped as described above and DNA was

ethanol-precipitated. The DNA precipitate was re-dis-

solved in 10 mM Tris-HCl (pH 8.0 at 25 �C). The DNA

cleavage products were analysed by 15 % PAGE in a TBE

buffer. The gels were visualized after staining with Sybr

Gold using a 312-nm UV transilluminator and pho-

tographed with a dedicated photographic filter. The previ-

ously constructed [13], custom 390 bp PCR fragment

containing a single TaqII recognition sequence 50-GAC

CGA-30 (?) was used for comparative titration experi-

ments. The complete cleavage of the PCR product should

yield 48 and 340 bp fragments. However, wt TaqII REase

is unable to cleave DNA completely.

REase activity in a DNA cleavage assay

DNA cleavage was performed in the presence and absence

of 50 lM SAM (cofactor) or SIN (cofactor analogue)

according to the previously standardized conditions in two

types of the reaction buffer: (i) in the previously established

reaction buffer, minimizing wt TaqII REase ‘star’ activity

(Buffer A: 40 mM Tris-HCl pH 8.0 at 70 �C, 10 mM

(NH4)2SO4, 10 mM MgCl2, 1 mM DTT, BSA 100 lg/ml]

[13], and (ii) for maximum N472A TaqII activity [Buffer B:

10 mM Tris-HCl pH 7.2 at 70 �C, 10 mM MgCl2, 1 mM

DTT, BSA 100 lg/ml) [25]. Two DNA substrates: pUC19

plasmid DNA, containing three TaqII recognition sequences

50-GACCGA-30 and the previously constructed, custom

390 bp PCR fragment [13] containing a single TaqII

recognition sequence 50-GACCGA-30 (?) were used for

comparative titration experiments. The reaction volume of

50 ll contained either 300 ng of the custom PCR substrate

or 500 ng of pUC19 DNA. All experiments utilized

recombinant wt RM.TaqII and the N472A RM.TaqII protein

variants. Titration experiments were carried out as series of

twofold dilutions, starting from 1250 ng (10 pmol) down to

19.5 ng (0.078 pmol) of both protein variants. After 1 h at

70 �C the reactions were immediately stopped by the addi-

tion of EDTA, SDS and proteinase K and the proteins were

digested for 1 h at 55 �C; phenol–chloroform extracted and

DNA was ethanol-precipitated. Such an extensive procedure

was necessary to remove TaqII protein from the reaction

mixture to ensure the proper migration of the resulting

restriction fragments during electrophoresis. The DNA was

re-dissolved in 10 mM Tris-HCl (pH 8.0 at 25 �C). The

DNA cleavage products were analysed through 15 % PAGE

or 1.2 % agarose gels in a TBE buffer. The gels were

visualized as above.

Results and discussion

N-MTases, transferring a methyl group to the cytosine-N4-

or adenine-N6-positions, contain nine conserved aa motifs

[26]. In this paper, a designed aa change concerned aspar-

agine (at position 472 in the TaqII polypeptide)—an evo-

lutionary conserved residue from the MTase catalytic site.

The N472 residue is located in motif IV of MTase (con-

sensus sequence: (S/N/D)PP(Y/F/W)) [26] and corresponds

to N473 from RM.TspGWI protein [20]. In M.TaqI, the

conserved N105 residue is responsible for hydrogen bond

formation with the 6-amino group of the substrate adenine,

which is rotated out of the DNA helix [27]. The second,

weaker hydrogen bond is formed between the adenine and

the first proline (P106) of the NPPY motif. The formed

hydrogen bonds increase the partial negative charge of the

N6 atom of the substrate nucleotide and activate it for direct

nucleophilic attack on the methyl group of the cofactor [27].

In this paper we substituted N472 with alanine—a small,

hydrophobic aa, which is unable to form the hydrogen bond

with the adenine. This substitution was originally designed

to eliminate TaqII MTase activity but instead it has caused a

complex, pleiotropic effect.

The aim of this study was to investigate the effect of this

particular aa substitution within the catalytic site of TaqII

MTase. For that purpose both wt RM.TaqII and the N472A

RM.TaqII variants were purified using the following

purification stages: heat treatment of bacterial lysates, ion

exchange chromatography, affinity chromatography and

size exclusion chromatography. The purity of both enzyme

preparations was estimated on a 10 % SDS-PAGE gel

(Fig. 1).

Bearing in mind the significant aa sequence similarity

between tspGWIRM and taqIIRM genes (64 % identity,

75 % positives, E value 0.0) [9, 15], as well as the identity

of MTase catalytic/SAM-binding motifs, we expected that

the corresponding TaqII and TspGWI protein variants

would exhibit similar biochemical features. However, we

found fundamental differences between them.

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Viability and morphological changes of recombinant

E. coli expressing mutated taqIIRM gene

Both recombinant wt RM.TaqII and N472A RM.TaqII

protein variants accumulated very slowly after induction of

the bacteriophage lambda PR promoter using a temperature

shift from 30 to 42 �C. However, in contrast to recombi-

nant E. coli [pRZ-wt-taqIIRM], E. coli [pRZ-taqIIRM-

N472A] cells exhibited a highly increased fragility after

induction. Therefore, in order to purify the N472A TaqII

protein variant, the culture had to be stopped no later than

2.5 h after induction.

Even barely detectable amounts of the N472A RM.TaqII

variant caused spontaneous bacterial cell lysis and a rapid

decrease in the OD600 value, measured 2–3 h after induction

of gene expression (Fig. 1a). Bacteria had to be immediately

centrifuged at room temperature, as pre-cooling of the bac-

terial culture resulted in rapid cell lysis. Cell weakness of the

taqIIRM-N472A mutant-carrying bacteria was associated with

an altered cell morphology (Fig. 2). Before induction, the

E. coli [pRZ-taqIIRM-N472A] cells growing at 30 �C were

noticeably longer than those expressing the wt taqIIRM gene

and tended to produce filaments of variable length (Fig. 2). A

similar effect, but to a lesser extent, was observed for

recombinant E. coli [pRZ-tspGWIRM-N473A] [20]. In com-

parison to N473A TspGWI producing cells, bacteria producing

the N472A RM.TaqII protein variant more visibly changed

their morphology. Approximately 1.5 h after induction of the

gene expression, the cells shortened and started to swell

(Fig. 2). 3 h after induction, a significant decrease in the

viable cell number was observed (Table 2) and the remaining

Fig. 1 Kinetics of E. coli cultures growth, harbouring pRZ-wt-

taqIIRM or E. pRZ-taqIIRM-N472A and analysis of purified RM.TaqII

proteins’ variants. A. Kinetics of E. coli [pRZ-wt-taqIIRM] (black

lines marked with circles or squares) and E. coli [pRZ-taqIIRM-

N472A] (red lines marked with x or triangles) bacterial culture

growth. The cultures were initially cultivated in TB media at 30 �C.

The cultures were induced by a temperature shift to 42 �C (solid

lines). The moment of induction is marked with an arrow. The control

cultures (dashed lines) were further cultivated at 30 �C without

induction. B. Purified protein preparations containing 300 ng of wt

TaqII or N472A TaqII protein were electrophoresed in 10 % SDS-

PAGE. Lane M1 Page Ruler Broad Range Protein Marker (Thermo

Scientific); lane M2 Unstained Protein MW Marker (Thermo

Scientific); lane 1 300 ng of wt recombinant TaqII REase; lane 2

300 ng of N472A TaqII variant. Both TaqII variants are indicated with

an arrow. (Color figure online)

Fig. 2 Microscope imaging of methylene blue stained E. coli cells

harbouring wt taqIIRM or taqIIRM N472A gene. Samples of the bacterial

culture were taken both from E. coli cells expressing the wt RM.TaqII

and N472A RM.TaqII variant, before promoter PR transcription induc-

tion, 1.5 and 3 h after induction. After methylene blue positive staining,

slide glass preparations were observed under an Olympus CX21FS1 light

microscope with 91200 magnification. (Color figure online)

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bacterial cells resembled those producing the wt TaqII protein

(Fig. 2). Further prolongation of culturing and the accumu-

lation of the ‘toxic’ N472A RM.TaqII protein variant

increased the selective pressure, giving an advantage to cells

that had lost the ability to produce the functional TaqII

REase. This probably resulted from alteration of the

recombinant pRZ-taqIIRM-N472A plasmid, while retaining

resistance to chloramphenicol (Fig. 1a). As neither N472A

TaqII REase activity nor the corresponding protein band on

SDS-PAGE were detectable in bacterial pellets starting from

4 h after induction (not shown), the observed lack of the

recombinant N472A TaqII variant was apparently caused by

random mutations in the taqIIRM gene or its associated

regulatory regions. We anticipate that the observed fila-

mentation and increased fragility of the cells might have been

caused by SOS response induction due to the impairment of

MTase activity and resulting DNA fragmentation [28, 29]. At

temperatures lower than 45 �C, both TaqII REase and MTase

exhibit only a fraction of their maximal activity at 70 �C[14]. Given the fact that at 42 �C the activity of the wt TaqII

REase is less than 0.6 % [14], while wt TaqII MTase shows

no significant drop in activity in comparison to its activity at

70 �C (not shown), it is not surprising that E. coli, harbouring

a wt recombinant taqIIRM gene, is able to grow despite the

enzyme overproduction.

The N472A TaqII MTase remains eightfold less active

than the wt recombinant TaqII MTase both at 42 and 70 �C(not shown). That leads to insufficient DNA protection

against the accumulating N472A TaqII REase, leading in

turn to rapid bacterial lysis, represented by an approx.

threefold decrease in the OD600 value measured 3 h after

induction compared with the induced, wt recombinant

RM.TaqII producing culture (Fig. 1a). As can be seen in

Fig. 1a, bacteria from the induced and non-induced control

cultures grew proportionally, reaching high optical density

values. The residual REase activity of the protein, devoid

of MTase protection, is apparently lethal for E. coli cells

due to unrepaired host genomic DNA cleavage.

Effect of N472A aa substitution on TaqII MTase

activity

Even though the RM.TaqII is a homologue of RM.TspGWI

(64 % identity, 75 % positives, E value 0.0) [15], the

analogous aa substitution in the TaqII MTase catalytic site

shows surprisingly different effects. In contrast to N473A

TspGWI, the introduced mutation in the taqIIRM gene

resulted in a protein variant with reduced, but not totally

eliminated REase and MTase activities.

A preliminary analysis of N472A TaqII MTase per-

formed by the cleavage protection assay proved that the

introduced aa change significantly impaired the investi-

gated enzyme activity. The wt recombinant TaqII MTase

(Fig. 3a) was estimated to be eightfold more active than the

N472A TaqII MTase (Fig. 3b). While the most precise

determinations of methylation activities can be achieved

using a standard assay measuring the introduction of the

radiolabelled methyl group into a substrate DNA, the used

DNA protection assay provides quantitative results, ade-

quate for the scope of this paper.

Interestingly, both investigated TaqII MTase variants

seem to methylate not only their cognate recognition

sequence, but also the degenerated ‘affinity star’ variants of

50-GACCGA-30. The reduction of the intensity of minor,

non-cognate bands can be noticed, which might be a con-

sequence of ‘affinity star’ DNA methylation (Fig. 3a, b). It

remains to be evaluated whether this ‘star’ MTase activity

is able to protect all the possible ‘affinity star’ variants of

the 50-GACCGA-30 DNA sequence with a preferred single-

bp departure from the canonical TaqII site [10].

Effect of N472A aa substitution on TaqII REase

Further evaluation of the biochemical properties of N472A

TaqII REase revealed another interesting feature of this variant

compared to the wt TaqII. The investigated N472A TaqII

REase activity appeared to be very strongly dependent on

changes in the reaction buffer, implying the important role of

the formation of particular ‘sensitive’ hydrogen bonds or

presence/absence of ‘salt bridges’. In fact, this feature is rather

outstanding compared to other enzymes, not only REases.

TaqII REase activity toward a single site DNA substrate

As seen in Fig. 4a and d, both wt TaqII and N472A TaqII

REase activities towards a short, single site PCR substrate

were barely detectable in the previously described stan-

dardized reaction buffer A in the absence of cofactor or its

analogue. Buffer A was established previously by our-

selves for minimizing wt TaqII* REase activity [13]. In

addition, the lack of the N472A TaqII REase activity was

also observed in titration experiments, performed in buffer

Table 2 Total number CFU (colony forming units) of recombinant E. coli 3 h after induction of the taqIIRM or taqIIRM-N472A gene expression

Culture medium Bacteria CFU (3 h after PR induction) OD600

TB E. coli [pRZ-wt-taqIIRM] 9.5 9 106 2.07

TB E. coli [pRZ-taqIIRM-N472A] 6.9 9 104 2.23

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A with longer DNA substrates (not shown). Moreover, nei-

ther SAM nor SIN could restore the N472A TaqII REase

activity under those conditions (Fig. 4e, f). To compare the

activity of the TaqII REase variants toward a single site DNA

substrate (between the experiments using different reaction

buffers), a well-defined reference point had to be selected.

For that purpose, we chose the band corresponding to the

48-bp cleavage product, obtained after cleavage with the wt

recombinant TaqII REase in buffer A without SAM or SIN

(Fig. 4a; lane 1, marked with an asterisk). This reference

point was used for all further comparisons and calculations.

In contrast to the N472A TaqII variant, wt TaqII REase

exhibited a 32-fold allosteric activation in the presence of

SAM and 64-fold activation in the presence of its analogue

SIN in reaction buffer A (Fig. 4b, c; Table 1). Both SAM and

SIN also stimulated our previously described [10] TaqII

REase ‘affinity star’ activity (Fig. 4b, c). The strongest TaqII

REase ‘affinity star’ activity appears in the presence of SIN

and can be further enhanced with DMSO [10]. The SIN-

induced relaxation of the TaqII DNA recognition sequence

allows for the recognition and cleavage of single-bp or 2-bp

departures from the canonical TaqII site, depending on the

presence of DMSO in the reaction buffer [10].

Interestingly, N472A TaqII REase activity could be effi-

ciently restored in the presence of SIN, while performing

cleavage reactions in a standardized buffer B of pH 7.2 [13,

25]. A very slight pH change from 8.0 to 7.2 and decrease of

ionic strength by removal of 10 mM (NH4)2SO4 from the

reaction buffer, resulted in significant restoration of N472A

TaqII REase activity towards a single site PCR substrate

(Fig. 5d–f; Table 1). In buffer B and in the absence of the

cofactor or SIN, both wt TaqII and N472A TaqII REase

activity were easily detectable, with wt TaqII REase approx.

16-fold and 128-fold more active in the presence of SAM and

SIN, respectively (Fig. 5b, c), when compared to the estab-

lished reference point. After the addition of SAM, the

investigated N472A TaqII REase activity in buffer B was

slightly increased in comparison to reactions devoid of this

cofactor (Fig. 5d, e). The best allosteric activation propen-

sity of both investigated TaqII REase variants in buffer B

towards a single site DNA substrate was observed in the

presence of SIN (Fig. 5c, f), with 32-fold enhancement of the

N472A TaqII REase activity and 128-fold allosteric activa-

tion of the wt TaqII REase. Remarkably, in contrast to the wt

recombinant RM.TaqII enzyme (Fig. 5c), the N472A variant

in the presence of SIN didn’t exhibit ‘affinity star’ relaxation

and yielded a more precise cleavage of the canonical TaqII

site, located within a 390 bp DNA fragment (Fig. 5f). This

suggests that, at the allosteric site, a delicate equilibrium is

reached between bound cofactor analogue SIN (which has

reversed charge distribution compared to SAM), hydrogen

bonding and ‘salt bridges’. The obtained equilibrium favours

the activity of both REase variants. However, the stimulation

level and enzyme fidelity is variable.

Fig. 3 Comparative titration of TaqII MTase protein variants in the

presence of SAM. 300 ng of 390 bp custom PCR, containing a single

recognition sequence (?) (1.2 pmol TaqII recognition sites), was

incubated with consecutive twofold MTase dilutions, starting from

1250 ng (10 pmol; 8.3:1 molar ratio of enzyme to recognition

sequence) down to 78 ng (0.31 pmol; 0.26:1 molar ratio of enzyme to

recognition sequence) of the wt RM.TaqII or N472A RM.TaqII

variants in the MTase buffer, supplemented with 200 lM of the SAM,

for 1 h, at 70 �C. Proteins were removed by proteinase K digestion.

The DNA was purified and challenged with an excess of the wt

RM.TaqII for 1 h at 70 �C in TaqII REase buffer supplemented with

20 lM SAM. Vertical arrows indicate lanes of the estimated identical

or nearly identical extent of DNA cleavage, horizontal arrows

indicate the position of a reference cleavage product (48 bp). a Lane

M GeneRulerTM 20 bp DNA Ladder (Thermo Scientific) (selected

bands marked); lane K untreated PCR DNA; lane 1–6 PCR DNA

incubated with the wt TaqII MTase (twofold serial dilutions),

subsequently cleaved with the wt RM.TaqII; lane 7, PCR DNA

cleaved with the RM.TaqII without previous incubation with the

enzyme in MTase buffer. b As in a, except that twofold serial

dilutions were performed with the N472A RM.TaqII variant

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TaqII REase activity toward DNA substrates with multiple

recognition sites

In order to further investigate the biochemical features of

the N472A RM.TaqII variant, a series of analogous exper-

iments using pUC19 plasmid DNA (three TaqII canonical

recognition sites) and bacteriophage lambda DNA (ten

TaqII canonical recognition sites) were performed. The

REase activity of the investigated TaqII variants toward

longer DNA molecules with multiple TaqII restriction sites

was evaluated (Figs. 6, 7).

For that purpose, analogous comparative titrations of

TaqII REase protein variants using pUC19 plasmid DNA

were performed as described for a single site DNA sub-

strate in the previous section. The complete TaqII cleavage

of pUC19 should result in three restriction fragments:

1337, 1160 and 183 bp (Fig. 7b). However, TaqII REase

belongs to a group of restriction enzymes that are unable to

cleave DNA completely. Thus, a stable partial cleavage

pattern was observed in most presented experiments

(Figs. 6, 7a). In buffer B and in the absence of the cofactor

or SIN, both wt RM.TaqII and N472A RM.TaqII REase

activity were detectable (Fig. 6a, d). However, the activity

of the N472A TaqII REase was significantly reduced in

comparison to the wt enzyme, as observed for a 390 bp

DNA substrate (Fig. 5a, d). After the addition of SAM, the

investigated N472A TaqII REase activity was significantly

increased (Fig. 6e) in comparison to reactions devoid of the

cofactor (Fig. 6d). The best allosteric activation propensity

of both investigated TaqII REase variants was observed in

the presence of SIN (Fig. 6c, f), similarly to the experi-

ments using a 390 bp DNA substrate (Fig. 5). Moreover, in

the presence of SIN both enzyme variants cleaved DNA to

completion (Fig. 6c, f). Remarkably, in contrast to wt

Fig. 4 Comparative titration of TaqII REase protein variants in the

presence or absence of SAM or SIN under conditions minimizing

TaqII* activity. 300 ng of 390 bp custom PCR, containing a single

TaqII recognition sequence (?) (1.2 pmol TaqII recognition sites),

was digested with consecutive twofold REase dilutions, starting from

1250 ng (10 pmol; 8.3:1 molar ratio of enzyme to recognition

sequence) down to 19.5 ng (0.078 pmol; 0.06:1 molar ratio of

enzyme to recognition sequence) of the wt RM.TaqII or N472A

RM.TaqII variants in buffer A developed for minimal TaqII* activity

[13], supplemented with 50 lM of the SAM or SIN, for 1 h, at 70 �C.

Vertical arrows indicate lanes of the estimated identical or nearly

identical extent of DNA cleavage, horizontal arrows indicate the

position of a reference cleavage product (48 bp). An asterisk indicate

a reference point for the comparison of wt TaqII and N472A TaqII

REase activity in buffers A and B. a Cleavage pattern of the wt

RM.TaqII in the absence of SAM and SIN. Lane M GeneRulerTM

20 bp DNA Ladder (Thermo Scientific) (selected bands marked); lane

K untreated PCR DNA; lane 1–8 PCR DNA cleaved with wt

RM.TaqII protein variant, twofold serial dilutions. The reaction

products were resolved on 15 % PAGE in TBE buffer and stained

with Sybr Gold. b As in a, except that the reactions were

supplemented with 50 lM of SAM. c As in a, except that the

reactions were supplemented with 50 lM of SIN. d As in a, except

that the reactions were carried out with the N472A RM.TaqII variant.

e As in a, except that the reactions were carried out with the N472A

RM.TaqII variant and supplemented with 50 lM of SAM. f As in a,

except that the reactions were carried out with the N472A RM.TaqII

variant and supplemented with 50 lM of SIN

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recombinant TaqII enzyme (Fig. 6c), the N472A variant in

the presence of SAM or SIN exhibited significantly reduced

‘affinity star’ relaxation and yielded a more precise cleavage

of the canonical TaqII sites, located within a pUC19 plasmid

DNA, as was the case for a single-site substrate (Fig. 6e, f).

A comparison of the obtained cleavage patterns is presented

in Fig. 7a. As is apparent from the presented experiments

and in contrast to wt TaqII, the N472A TaqII REase can

cleave only a small fraction of the possible degenerated

variants of canonical TaqII recognition sequence, possibly

those with only a single degeneracy per variant allowed.

We have demonstrated in our previous papers that the

TspGWI and TaqII REase ‘affinity star’ activity is strongly

stimulated by the presence of the cognate recognition sequence

within the cleaved DNA molecule [10, 30]. Although the

investigated enzyme variants are able to cleave DNA sub-

strates with a single recognition site, they seem to be stimulated

by the presence of two cognate recognition sequences located

in a cis configuration within the DNA molecule. We hypoth-

esize that the degenerated recognition sequence variant located

in the cis configuration could substitute the second, cognate

TaqII site, stimulating the enzyme to efficient DNA cleavage.

Remarkably, the introduced N472A aa change significantly

reduced the ability of N472A RM.TaqII REase to recognize and

cleave the degenerated TaqII recognition sequences, increas-

ing the enzyme fidelity toward the cognate recognition

sequence. This effect is particularly noticeable in the presence

of SIN (Figs. 6f, 7a). The intensity of the bands corresponding

to restriction fragments 1337 bp and 1160 bp seem to be

similar, despite different amounts of N472A RM.TaqII in the

reaction mixture (Fig. 6f). However, in the case of the wt TaqII

REase, the band corresponding to the 1337 bp DNA fragment

visibly changes its intensity, depending on the concentration of

the enzyme (Fig. 6c). In contrast to the 1160 bp DNA

Fig. 5 Comparative titration of TaqII REase protein variants in the

presence or absence of SAM or SIN under conditions maximizing

N472A TaqII activity. 300 ng of 390 bp custom PCR, containing a

single recognition sequence (?) (1.2 pmol TaqII recognition sites),

was digested with consecutive twofold REase dilutions, starting from

1250 ng (10 pmol; 8.3:1 molar ratio of enzyme to recognition

sequence) down to 19.5 ng (0.078 pmol; 0.06:1 molar ratio of

enzyme to recognition sequence) of the wt RM.TaqII or N472A

RM.TaqII variants in REase buffer B, supplemented with 50 lM of

SAM or SIN, for 1 h, at 70 �C. Vertical arrows indicate lanes of the

estimated identical or nearly identical extent of DNA cleavage,

horizontal arrows indicate the position of a reference cleavage

product (48 bp). A reference point used for the comparison of

recombinant wt and N472A TaqII REase activity is marked with an

asterisk (Fig. 4a, lane 1). a Cleavage pattern of the wt TaqII REase in

the absence of SAM and SIN. Lane M GeneRulerTM 20 bp DNA

Ladder (Thermo Scientific) (selected bands marked); lane K untreated

PCR DNA; lane 1-8 PCR DNA cleaved with the wt RM.TaqII protein

variant, twofold serial dilutions. The reaction products were resolved

on 15 % polyacrylamide gel in TBE buffer and stained with Sybr

Gold. b As in a, except that the reactions were supplemented with

50 lM of SAM. c As in a, except that the reactions were

supplemented with 50 lM of SIN. d As in a, except that the

reactions were carried out with the N472A RM.TaqII variant. e As in

a, except that the reactions were carried out with the N472A RM.TaqII

variant and supplemented with 50 lM of SAM. f. As in a, except that

the reactions were carried out with the N472A RM.TaqII variant and

supplemented with 50 lM of SIN

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fragment, the 1337 bp restriction fragment contains a single

cognate 50-GACCGA-30 recognition sequence, which proba-

bly stimulates cleavage of the degenerated sequence variants.

Such stimulation seems to be present only in the case of the wt

TaqII REase (Fig. 6c, f). The intensity of the second band

corresponding to the 1160 bp DNA fragment (devoid of the

cognate 50-GACCGA-30 sequence) remains stable, irrespec-

tive of the TaqII variant used.

All the observations discussed above suggest that the

difference between both RM.TaqII variants is rather in the

allosteric activation propensity. Such a radical increase in

the allosteric activation sensitivity, caused by a minor pH

shift of 0.8 units close to the cytoplasmic physiological

range and minimal ionic strength decrease, is an unexpected

phenomenon as the wt RM.TaqII exhibits over 80 % activity

within this pH and ionic strength range [13]. We further

hypothesize that the N472A aa substitution within RM.TaqII

causes a ‘wobble’ tertiary structure of the protein, which

needs a stabilizing aid in the form of crucial ‘salt bridges’.

The ‘salt bridges’ are highly affected by a minor pH change.

A ‘molecular clip’ in the form of hydrogen bonding and the

presence of the cofactor analogue SIN is necessary for

restoration of the N472A REase activity. It is also possible

that, as a result of the N472A aa change, the TaqII enzyme

exhibits weaker or impaired specific DNA binding in com-

parison to the wt recombinant protein. The SIN-bound state

of the N472A RM.TaqII variant—with its conformation

favouring a pathway for cleavage—could almost neutralize

the effect of the introduced mutation, restoring efficient,

specific protein-DNA binding and DNA cleavage.

Summarizing, the introduced mutation had several effects

on the properties and activities of the engineered N472A

RM.TaqII enzyme: (i) higher toxicity to recombinant E. coli

(Figs. 1a, 2); (ii) partial reduction of MTase activity

(Fig. 3); (iii) partial reduction of REase activity (Figs. 4, 5,

6); (iv) enormous REase sensitivity to very minor pH and

salt concentration differences, which act as a 0/1 ‘switch’

(Figs. 4, 5); (v) different effects of SAM and SIN: the for-

mer only partially reactivated REase activity, while the latter

caused a significant REase allosteric activation (Figs. 4, 5,

6); (vi) increase in DNA cleavage fidelity, exceeding that of

wt RM.TaqII (Table 1; Figs. 5f, 7) [10, 13].

As DNA recognition fidelity of the N472A RM.TaqII

variant is significantly increased, compared to the wt

recombinant RM.TaqII, the N472A TaqII REase may be

considered a novel enzyme—an improved version toward a

more independent enzymatic action. Such an improved

enzyme version has a higher potential applicability in

genetic engineering technologies.

Our novel method for the construction of useful protein

variants of the bifunctional REases–MTases through a

simple aa substitution within the NPPY motif seems to be

Fig. 6 Comparative titration of TaqII REase protein variants using

pUC19 plasmid DNA. 500 ng of pUC19 plasmid DNA was digested

with consecutive twofold REase dilutions, starting from 416 ng

(3.31 pmol; 4:1 molar ratio of enzyme to recognition sequence) down

to 1625 ng (12,9 fmol; 0.0016:1 molar ratio of enzyme to recognition

sequence) of the wt RM.TaqII or N472A RM.TaqII variants in REase

buffer B, supplemented with 50 lM of SAM or SIN, for 1 h, at 70 �C.

a Cleavage pattern of the wt RM.TaqII in the absence of SAM and

SIN. Lane M1, GeneRulerTM 1 kb DNA Ladder (Thermo Scientific)

(selected bands marked); lane M2, GeneRulerTM 100 bp DNA Ladder

(Thermo Scientific) (selected bands marked); lane K untreated pUC19

DNA; lane 1–9 DNA cleaved with the wt RM.TaqII protein variant,

twofold serial dilutions. The reaction products were resolved on

1.2 % agarose gel in TBE buffer and stained with ethidium bromide.

b As in a, except that the reactions were supplemented with 50 lM of

SAM. c As in a, except that the reactions were supplemented with

50 lM of SIN. d As in a, except that the reactions were carried out

with the N472A RM.TaqII variant. e As in a, except that the reactions

were carried out with the N472A RM.TaqII variant and supplemented

with 50 lM of SAM. f As in a, except that the reactions were carried

out with the N472A RM.TaqII variant and supplemented with 50 lM

of SIN

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of general application, and can be used for the other

enzymes of this type. So far we have successfully tested

this method for RM.TaqII and RM.TthHB27I (manuscripts

in preparation). Possibly, introducing other aa with differ-

ent side chain properties at this particular position may

result in enhancement and/or modulation of both enzymatic

activities, leading to the generation of useful protein vari-

ants, not only with improved fidelity but also increased

specific activity and ability to complete DNA cleavage.

Conclusions

(i) The taqIIRM gene from T. aquaticus YT-1 was

subjected to site directed mutagenesis, resulting in

a N472A substitution within the NPPY motif of

TaqII prototype REase–MTase. The mutated gene

was expressed in E. coli and the N472A protein

variant was isolated.

(ii) Methylation activity of N472A RM.TaqII variant

towards cognate DNA is significantly decreased.

In vivo this is manifested as a strong ‘toxic’ effect

to the E. coli host, indicating inadequate host

genomic DNA protection by inefficient MTase

activity.

(iii) The N472A TaqII REase activity strongly depends

on pH, salt concentration and the presence of

SAM or SIN.

(iv) An addition of the cofactor analogue SIN results

in the allosteric activation of the N472A TaqII

REase toward a single site DNA substrate,

reaching approx. 25–50 % of the wt TaqII

activity.

Fig. 7 Cleavage of longer

DNA substrates using wt and

N472A RM.TaqII protein

variants. The reaction products

were resolved in 1.2 % agarose

gel in TBE buffer and stained

with ethidium bromide.

a 500 ng of pUC19 plasmid

DNA was cleaved with TaqII

REase variants (2:1 molar ratio

of enzyme to recognition

sequence) in REase buffer B,

supplemented with 50 lM of

SIN, for 1 h, at 70 �C. Lane M1

GeneRulerTM 100 bp DNA

Ladder (Thermo Scientific)

(selected bands marked); lane K

untreated pUC19 DNA; lane 1

DNA cleaved with the wt

RM.TaqII protein; lane 2 DNA

cleaved with the N472A

RM.TaqII variant. b The

predicted TaqII cleavage pattern

of pUC19 plasmid DNA. Lane L

linear form of pUC19 plasmid

DNA. c 500 ng of lambda DNA

was cleaved with TaqII REase

variants (2:1 molar ratio of

enzyme to recognition

sequence) in REase buffer B,

supplemented with 50 lM of

SIN, for 1 h, at 70 �C. Lane M2

GeneRulerTM 1 kb DNA Ladder

(Thermo Scientific) (selected

bands marked); lane K

untreated lambda DNA; lane 1

DNA cleaved with the wt

RM.TaqII protein; lane 2 DNA

cleaved with the N472A

RM.TaqII variant. d The

predicted TaqII cleavage pattern

of bacteriophage lambda DNA

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(v) In comparison to wt TaqII REase, the N472A TaqII

variant exhibits significantly reduced ‘affinity star’

relaxation towards very frequent cleavage and

increased fidelity towards a cognate 6-bp site

recognition sequence.

(vi) A novel method of rational engineering of the IIS/

IIC/IIG REases’ fidelity/activity was further

developed.

Acknowledgments We thank Patrick Groves and Joanna Jezewska-

Frackowiak for the proof-reading of the manuscript. This work was

supported by The Polish Ministry of Science and Higher Education

grant DS/530-8640-D509-14, BMN 538-8645-B665-14 and

538-8645-B655-15 (University of Gdansk, Chemistry Faculty,

Molecular Biotechnology Department) and in part by BioVentures

Institute Ltd. (Poznan, Poland).

Author Contribution AZS conceived and coordinated the project,

designed experiments, prepared the figures and drafted the manu-

script. PMS came up with the concept of the Thermus sp. enzymes

family, consulted the interpretation of experimental data analysis and

critically read the manuscript. JZ optimized gene expression condi-

tions, established a procedure for N472A TaqII isolation and per-

formed analyses of MTase activity. EC and ES performed a

biochemical characteristic of the TaqII REase variants. WW per-

formed mutagenesis and preliminary expression experiments. All

authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest The authors declare that they have no com-

peting interest.

References

1. Gupta D, Sharma N (2014) Thermostable restriction endonucle-

ases from thermophilic bacteria. Int Res J Pharm 5(4):259–

263. doi:10.7897/2230-8407

2. Wang X, Gou D, Xu SY (2010) Polymerase-endonuclease

amplification reaction (PEAR) for large-scale enzymatic pro-

duction of antisense oligonucleotides. PLoS One 5(1):e84

30. doi:10.1371/journal.pone.0008430

3. Huang CG, Agre P, Strange K, Lamitina T (2006) Isolation of C.

elegans deletion mutants following ENU mutagenesis and ther-

mostable restriction enzyme PCR screening. Mol Biotechnol

32(1):83–86

4. Zhao J, Xie F, Zhong W, Wu W, Qu S, Gao S, Liu L, Zhao J,

Wang M, Zhou J, Jie H, Chen W (2013) Restriction endonuclease-

mediated real-time digestion-PCR for somatic mutation detection.

Int J Cancer 132(12):2858–2866. doi:10.1002/ijc.27968

5. Liang J, Blumenthal RM (2013) Naturally-occurring, dually-

functional fusions between restriction endonucleases and regu-

latory proteins. BMC Evol Biol 13:218. doi:10.1186/1471-2148-

13-218

6. Zylicz-Stachula A, Harasimowicz-Slowinska RI, Sobolewski I,

Skowron PM (2002) TspGWI, a thermophilic class-IIS restriction

endonuclease from Thermus sp., recognizes novel asymmetric

sequence 50-ACGGA(N11/9)-30. Nucleic Acids Res 30:e33

7. Skowron PM, Majewski J, Zylicz-Stachula A, Rutkowska SM,

Jaworowska I, Harasimowicz-Slowinska RI (2003) A new

Thermus sp. class-IIS enzyme sub-family: isolation of a ‘twin’

endonuclease TspDTI with a novel specificity 50-ATGAA(N(11/

9))-30, related to TspGWI, TaqII and Tth111II. Nucleic Acids Res

31:e74

8. Loenen WA, Dryden DT, Raleigh EA, Wilson GG (2014) Type I

restriction enzymes and their relatives. Nucleic Acids Res

42(1):20–44. doi:10.1093/nar/gkt847

9. Zylicz-Stachula A, Bujnicki JM, Skowron PM (2009) Cloning and

analysis of bifunctional DNA methyltransferase/nuclease TspGWI,

the prototype of a Thermus sp. family. BMC Mol Biol 10:52. doi:10.

1186/1471-2199-10-52

10. Zylicz-Stachula A, Zolnierkiewicz O, Jasiecki J, Skowron PM

(2013) A new genomic tool, ultra-frequently cleaving TaqII/

sinefungin endonuclease with a combined 2.9-bp recognition site,

applied to the construction of horse DNA libraries. BMC Genom

14(1):370. doi:10.1186/1471-2164-14-370

11. Zylicz-Stachula A, Zolnierkiewicz O, Lubys A, Ramanauskaite

D, Mitkaite G, Bujnicki JM, Skowron PM (2012) Related

bifunctional restriction endonuclease-methyltransferase triplets:

TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities.

BMC Mol Biol 13:13. doi:10.1186/1471-2199-13-13

12. Barker D, Hoff M, Oliphant A, White R (1984) A second type II

restriction endonuclease from Thermus aquaticus with unusual

sequence specificity. Nucleic Acids Res 12:5567–5581

13. Zylicz-Stachula A, Zolnierkiewicz O, Sliwinska K, Jezewska-

Frackowiak J, Skowron PM (2011) Bifunctional TaqII restriction

endonuclease: redefining the prototype DNA recognition site and

establishing the Fidelity Index for partial cleaving. BMC Bio-

chem 12:62. doi:10.1186/471-2091-12-62

14. Zylicz-Stachula A, Zolnierkiewicz O, Sliwinska K, Jezewska-

Frackowiak J, Skowron PM (2014) Modified ‘one amino acid-one

codon’ engineering of high GC content TaqII-coding gene from

thermophilic Thermus aquaticus results in radical expression

increase. Microb Cell Fact 13:7. doi:10.1186/1475-2859-13-7

15. Zylicz-Stachula A, Bujnicki JM, Skowron PM (2014) Erratum to:

cloning and analysis of a bifunctional methyltransferase/restriction

endonuclease TspGWI, the prototype of a Thermus sp. enzyme

family. BMC Mol Biol 15:16. doi:10.1186/1471-2199-15-16

16. Skowron PM, Vitkute J, Ramanauskaite D, Mitkaite G,

Jezewska-Frackowiak J, Zebrowska J, Zylicz-Stachula A, Lubys

A (2013) Three-stage biochemical selection: cloning of prototype

class IIS/IIC/IIG restriction endonuclease-methyltransferase TsoI

from the thermophile Thermus scotoductus. BMC Mol Biol

14:17. doi:10.1186/1471-2199-14-17

17. Shinomiya T, Kobayashi M, Sato S (1980) A second site specific

endonuclease from Thermus thermophilus 111, Tth111II. Nucleic

Acids Res 8(15):3275–3285

18. Zhu Z, Guan S, Robinson D, El Fezzazi H, Quimby A, Xu SY

(2014) Characterization of cleavage intermediate and star sites of

RM. Tth111II. Sci Rep 4:3838. doi:10.1038/serp03838

19. Krefft D, Zylicz-Stachula A, Mulkiewicz E, Papkov A, Jezewska-

Frackowiak J, Skowron PM (2015) Two-stage gene assem-

bly/cloning of a member of the TspDTI subfamily of bifunctional

restriction endonucleases, TthHB27I. J Biotechnol 194:67–80.

doi:10.1016/j.biotec2014.11.030

20. Zylicz-Stachula A, Jezewska-Frackowiak J, Skowron PM (2014)

Cofactor analogue-induced chemical reactivation of endonuclease

activity in a DNA cleavage/methylation deficient TspGWI N473A

variant in the NPPY motif. Mol Biol Rep 41(4):2313–2323. doi:10.

1007/s11033-014-3085-x

21. Wang H, Sivonen K, Rouhiainen L, Fewer DP, Lyra C, Rantala-

Ylinen A, Vestola J, Jokela J, Rantasarkka K, Li Z, Liu B (2012)

Genome-derived insights into the biology of the hepatotoxic

bloom-forming cyanobacterium Anabaena sp. strain 90. BMC

Genom 13(1):613. doi:10.1186/1471-2164-13-613

22. Sarrade-Loucheur A, Shuang-y X, Siu-Hong C (2013) The role of

the methyltransferase domain of bifunctional restriction enzyme

Mol Biol Rep

123

Author's personal copy

RM. BpuSI in cleavage activity. PLoS ONE 8(11):e80967.

doi:10.1371/journal.pone.0080967

23. Liebert K, Horton JR, Chahar S, Orwick M, Cheng X, Jeltsch A

(2007) Two alternative conformations of S-adenosyl-L-homo-

cysteine bound to Escherichia coli DNA adenine methyltrans-

ferase and the implication of conformational changes in

regulating the catalytic cycle. J Biol Chem 282(31):22848–22855

24. Green MR, Sambrook J (2012) Molecular cloning: a laboratory

manual, 4th edn. Cold Spring Harbor Laboratory Press, CSH,

New York

25. Byczk W (2012) Investigation of the effect of N472A amino acid

change in motif IV of methyltransferase on the TaqII enzyme

activity. Master Thesis, Department of Chemistry, University of

Gdansk

26. Malone T, Blumenthal RM, Cheng X (1995) Structure-guided

analysis reveals nine sequence motifs conserved among DNA

amino-methyltransferases, and suggests a catalytic mechanism

for these enzymes. J Mol Biol 253(4):618–632

27. Goedecke K, Pignot M, Goody RS, Scheidig AJ, Weinhold E

(2001) Structure of the N6-adenine DNA methyltransferase

M.TaqI in complex with DNA and a cofactor analog. Nat Struct

Biol 8(2):121–125

28. Bos J, Zhang Q, Vyawahare S, Rogers E, Rosenberg SM, Austin

RH (2015) Emergence of antibiotic resistance from multinucle-

ated bacterial filaments. Proc Natl Acad Sci USA 112(1):178–

183. doi:10.1073/pnas.1420702111

29. Erental A, Sharon I, Engelberg-Kulka H (2012) Two pro-

grammed cell death systems in Escherichia coli: an apoptotic-like

death is inhibited by the mazEF-mediated death pathway. PLoS

Biol 10(3):e1001281. doi:10.1371/journal.pbio.1001281

30. Zylicz-Stachula A, Zołnierkiewicz O, Je _zewska-Frackowiak J,

Skowron PM (2011) Chemically-induced affinity star restriction

specificity: a novel TspGWI/sinefungin endonuclease with theo-

retical 3-bp cleavage frequency. Biotechniques 50(6):397–

406. doi:10.2144/000113685

Mol Biol Rep

123

Author's personal copy