Structural studies of restriction-modification systems as a model … · 2019. 9. 10. · 5....

Honorata Czapińska

Structural studies of restriction-modification systems

as a model of protein-DNA interaction

Review of scientific achievements presented in the application

for the habilitation degree

(Autoreferat – English version)

Warsaw, 29.04.2019

Dr Honorata Czapińska Review of achievements

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1. Name, Surname:

Honorata Czapińska

2. Scientific degrees and titles:

23.06.1997 BSc in Biotechnology, University of Wrocław

25.05.1999 MSc in Biotechnology, University of Wrocław

08.10.2004 PhD in Biochemistry, University of Wrocław

PhD thesis title: Crystallographic analysis of bovine pancreatic trypsin inhibitor

(BPTI) mutants and their complexes with bovine chymotrypsin

3. Professional experience:

1999 - 2004 PhD student at the Institute of Biochemistry and Molecular

Biology, University of Wrocław

2004 - 2015 Postdoc at the International Institute of Molecular and Cell Biology

2015 - 2016 Postdoc at the Institute of Biochemistry and Biophysics PAS

2017 - present Senior Researcher at the International Institute of Molecular and

Cell Biology


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4. Scientific achievement referred to in Art. 16(2) of the Act of 14 March 2003 on scientific degrees and

titles and degrees and titles in arts (Journal of Laws of the Republic of Poland no. 65, item 595 amended)

a) Title of the scientific achievement

Structural studies of restriction-modification systems as a model of protein-DNA interaction

b) List of publications constituting the scientific achievement

1. Czapinska H, Siwek W, Szczepanowski RH, Bujnicki JM, Bochtler M, Skowronek KJ (2019) Crystal

structure and directed evolution of specificity of NlaIV restriction endonuclease. J Mol Biol

doi: 10.1016/j.jmb.2019.04.010.

2. Czapinska H, Kowalska M, Zagorskaite E, Manakova E, Slyvka A, Xu SY, Siksnys V, Sasnauskas G,

Bochtler M (2018) Activity and structure of EcoKMcrA. Nucleic Acids Res 46, 9829-41.

3. Mierzejewska K, Bochtler M$, Czapinska H$ (2016) On the role of steric clashes in methylation

control of restriction endonuclease activity. Nucleic Acids Res 44, 485-95.

4. Mierzejewska K*, Siwek W*, Czapinska H*, Kaus-Drobek M, Radlinska M, Skowronek K, Bujnicki JM,

Dadlez M, Bochtler M (2014) Structural basis of the methylation specificity of R.DpnI. Nucleic Acids

Res 42, 8745-54.

5. Kisiala M, Copelas A, Czapinska H, Xu SY, Bochtler M (2018) Crystal structure of the modification-

dependent SRA-HNH endonuclease TagI. Nucleic Acids Res 46, 10489-503.

6. Kazrani AA, Kowalska M, Czapinska H, Bochtler M (2014) Crystal structure of the 5hmC specific

endonuclease PvuRts1I. Nucleic Acids Res 42, 5929-36.

7. Wojciechowski M, Czapinska H, Bochtler M (2013) CpG underrepresentation and the bacterial

CpG-specific DNA methyltransferase M.MpeI. Proc Natl Acad Sci USA 110, 105-10.

8. Siwek W, Czapinska H, Bochtler M, Bujnicki JM, Skowronek K (2012) Crystal structure and

mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI.

Nucleic Acids Res 40, 7563-72.

9. Sokolowska M, Czapinska H, Bochtler M (2011) Hpy188I-DNA pre- and post-cleavage complexes -

snapshots of the GIY-YIG nuclease mediated catalysis. Nucleic Acids Res 39, 1554-64.

10. Firczuk M, Wojciechowski M, Czapinska H, Bochtler M (2011) DNA intercalation without flipping

in the specific ThaI-DNA complex. Nucleic Acids Res 39, 744-54.

$ Corresponding authors * Equal contributions


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11. Sokolowska M, Czapinska H, Bochtler M (2009) Crystal structure of the ββα-Me restriction

endonuclease Hpy99I. Nucleic Acids Res 37, 3799-810.

12. Szczepanowski RH, Carpenter MA, Czapinska H, Zaremba M, Tamulaitis G, Siksnys V, Bhagwat AS,

Bochtler M (2008) Central base pair flipping and discrimination by PspGI. Nucleic Acids Res 36,

6109-17.

13. Sokolowska M, Kaus-Drobek M, Czapinska H, Tamulaitis G, Siksnys V, Bochtler M (2007) Restriction

endonucleases that resemble a component of the bacterial DNA repair machinery. Cell Mol Life Sci

64, 2351-57.

14. Kaus-Drobek M, Czapinska H, Sokolowska M, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V,

Bochtler M (2007) Restriction endonuclease MvaI is a monomer that recognizes its target sequence

asymmetrically. Nucleic Acids Res 35, 2035-46.

15. Sokolowska M, Kaus-Drobek M, Czapinska H, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V,

Bochtler M (2007) Monomeric restriction endonuclease BcnI in the apo form and in an asymmetric

complex with target DNA. J Mol Biol 369, 722-34.

16. Bochtler M, Szczepanowski RH, Tamulaitis G, Grazulis S, Czapinska H, Manakova E, Siksnys V (2006)

Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. EMBO Journal 25,

2219-29.


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c) Review of the scientific objective and results obtained in the presented publications and their

potential application

Introduction

Restriction-modification (RM) systems play an important role in prokaryotic organisms. They constitute

one of the key bacterial defense lines (Doron et al., 2018). The RM systems use a simple mechanism of

distinguishing between own and alien DNA based on their different modification patterns. In a standard

arrangement, the host methylates its own DNA with the help of a sequence specific methyltransferase

(M), and destroys any incoming DNA that lacks the modification using a restriction endonuclease (R)

of matching specificity. However, some phages acquired the ability to arrange for the modification

of their DNA, so that it becomes invisible to a particular bacterial RM system. In turn, some hosts leave

their DNA non-methylated and use modification dependent enzymes to break up the foreign genetic

material (Loenen and Raleigh, 2014).

The endonucleases of restriction modification systems have found many applications as genetic

engineering tools. Sequence specific Type II restriction endonucleases that cleave non-modified DNA

are used to prepare DNA fragments for molecular cloning (Roberts, 2005). The modification specific

restriction endonuclease DpnI is applied for site directed mutagenesis, to selectively cut

the 6-methyladenine marked DNA template without damage to newly synthesized DNA (Liu and

Naismith, 2008). Pairs of CpG methylation sensitive and tolerant enzymes, such as MspI and HpaII,

are used to detect modification patterns in eukaryotic DNA (Yaish et al., 2014). Cytosine

hydroxymethylation dependent enzymes, such as PvuRts1I or AbaSI, are increasingly used to study the

role of this modification in eukaryotes (Wang et al., 2011; Sun et al., 2013). Sequence non-specific

catalytic domains of restriction endonucleases (particularly FokI) have been adapted to create "designer"

endonucleases, such as zinc finger nucleases (ZFNs) or transcription activator like nucleases (TALENs)

that could address a predetermined target site in the genome of interest before CRISPR-Cas systems

came to a wide use (Chandrasegaran and Carroll, 2016).

Aim of the studies

Our studies were aimed at the structural characterization of the interaction between restriction-

modification systems and their target DNA. The idea behind the project was to use the knowledge

obtained with the help of this simple but efficient model system to learn more about the rules governing

DNA recognition, cleavage and modification. Restriction endonucleases are particularly useful for this

purpose, primarily due to their very stringent selectivity for target DNA. Moreover, this group

of enzymes is extremely diverse and allows to study different means of DNA recognition and hydrolysis.

Methyltransferase components of the RM systems are much more conserved with respect to both fold

as well as catalytic mechanism (Cheng, 1995). In this case our goal was to find and characterize

a prokaryotic enzyme that would exhibit the CpG specificity, analogous to the eukaryotic Dnmt DNA

methyltransferases, structures of which were unknown at the time. We believed, that

a structure of such enzyme might shed light on the target sequence recognition mechanism of its

eukaryotic counterparts.


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Results

The first structural studies of restriction-modification systems were published at the end of the XXth

century and comprised EcoRI, EcoRV, M.HhaI, M.HaeIII and their DNA complexes (McClarin et al., 1986;

Kim et al., 1990; Winkler et al., 1993; Cheng et al., 1994; Klimasauskas et al., 1994; Reinisch et al., 1995).

Since then a number of structures have been determined (the state of art at the onset of our studies

is best reflected in the Pingoud et al. (2005) and Gromova and Khoroshaev (2003) review articles).

However, in case of the restriction endonucleases, the published structures covered only one catalytic

type (PD-(D/E)XK). We were interested in the classes of these enzymes that were yet uncharacterized

or exhibited unusual properties. We focused on the Type II enzymes, that cleave DNA within or in close

proximity to short target sequences. The relatively simple domain organization of these enzymes greatly

facilitates the structure-activity relationship studies. We were first guided by the cleavage stagger, which

inevitably correlates with the separation of the enzyme active sites and often dictates the endonuclease

properties (oligomerization state and/or domain organization). It also proved a great selection tool in the

search for unusual enzymes or members of new catalytic classes. Once most of the enzymes of different

cleavage staggers were characterized by us and others in the field, we have switched to studies

of unusual endonucleases that display specificity towards modified target sites.

Our search for a CpG specific prokaryotic methyltransferase was guided by the fact that the methylation

of cytosine residues leads to their increased deamination (Shen et al., 1994). This process is known to be

mutagenic and in a longer evolutionary perspective leads to the elimination of the methyltransferase

target sequences from the genome of its host (Shen et al., 1992). We have searched the available

prokaryotic genomes for the highest level of the CpG sequence depletion and then the proteomes of the

top scorers for the presence of an active C5-cytosine methyltransferase.

During our studies of restriction modification systems we have determined and analyzed 20 restriction

enzyme structures (of 12 distinct proteins) and one methyltransferase structure. The results and

implications of our studies are presented below with specific enzymes provided as illustrating examples.

The review is focused on the structural and bioinformatic part of the study, since this was my

contribution to the presented results.

I. Folds and catalytic mechanisms

Methyltransferases

The catalytic domains of DNA methyltransferases share a conserved core usually termed AdoMet-

dependent MTase fold (Cheng and Roberts, 2001). They all use S-adenosylmethionine as a methyl group

donor. In the case of M.MpeI and Dnmt1 the group is transferred onto the C5 atom of a cytosine

in a reaction that involves the formation of a covalent bond between an active site cysteine and

the cytosine base in the transition state (Cheng, 1995). However, there was still a controversy about the

details of the reaction mechanism - it can either proceed as a one or two step addition (Chen at al., 1993;

Gabbara et al., 1995). The determination of the M.MpeI-DNA complex structure to a relatively high

resolution was a first step of a project aiming at elucidation of this ambiguity (Wojciechowski, Czapinska,

Bochtler, 2013). Our crystals of M.MpeI-DNA complex diffracted to a resolution higher than previously

reported for any methyltransferase-DNA complex according to the Protein Data Bank. The good quality


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of the crystals makes it possible to obtain structures of M.MpeI complexes with various

substrate/product analogues and inhibitors. The details of the obtained structures should have

implications for the eukaryotic DNA methyltransferases. These are of interest as drug targets in human

malignancies (Saldívar-González et al., 2018; Lyko, 2018; Gnyszka et al., 2013; Siedlecki and

Zielenkiewicz, 2006), but harder to crystallize and less tractable than the prokaryotic models.

Endonucleases

Known restriction endonucleases belong to five distinct catalytic fold classes: PD-(D/E)XK, HNH (ββα-

Me), GIY-YIG, phospholipase D like and halfpipe (Orlowski and Bujnicki, 2008). The LAGLIDADG catalytic

motif was observed for homing endonucleases (Chevalier et al., 2005), but so far not confirmed

for REases. We have studied the members of PD-(D/E)XK, HNH and GIY-YIG families. Enzymes of the

three classes differ in fold but uniformly use metals as cofactors for the cleavage reaction and leave

5'-phosphorylated ends. They all use general base catalysis to activate the nucleophilic water that gets

incorporated into the product in a single substitution reaction. However, they differ with respect

to the nature of the base, number of metal ions in the active site and stringency of the requirement for

the metal ion identity. PD-(D/E)XK enzymes usually use an active site lysine as a base and perform

catalysis almost exclusively in the presence of magnesium ions. Two metal ions are typically found

in their active sites although there are reports in the literature indicating that this number may vary

(Horton et al., 1998, 2004). HNH endonucleases usually use a histidine residue to activate a water

molecule and a single ion of either magnesium or manganese for catalysis. The role of the general base

in GIY-YIG nucleases is played by a tyrosine most likely occurring in a phenolate form. This group

of enzymes is known to be active in the presence of a various divalent metal ions. As in the case of HNH

endonucleases, the active site of GIY-YIG nucleases hosts a single metal ion, which promotes

the departure of the leaving group.

Our studies of restriction endonucleases contributed substantially to the understanding of the catalytic

mechanisms of this group of enzymes. Back to back with the group of Prof. Barry Stoddard,

we have published the first DNA complex structures of the HNH and GIY-YIG restriction endonucleases

(Sokolowska, Czapinska, Bochtler, 2009; Sokolowska, Czapinska, Bochtler, 2011; Shen et al., 2010;

Mak et al., 2010). For the GIY-YIG nuclease we have described both the substrate and the product

complex. This was particularly interesting since the structure of no other DNA complex of a member

of this class was known before. The parallel work of our and Prof. Stoddard’s group on GIY-YIG nucleases

made it possible to describe their catalytic mechanism and model the complexes of the eukaryotic

enzymes. Our studies find further use in the analysis of new structures of these enzymes, as e.g. SLX1-

SLX4 endonuclease (Gaur et al., 2015).

II. DNA recognition

Hydrogen bonding interactions

Very stringent substrate selectivity of RM systems is crucial for the maintenance of the host genome

integrity. Any unspecific activity of the restriction endonuclease that is not matched by a protective

action of the partner methyltransferase is lethal for bacteria. This can easily be noticed while trying

to express only the nuclease component of the RM pair in a heterologous expression system.


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Such stringency is achieved by the RM systems in different ways. There are a few direct means to read

the DNA sequence. The most pronounced of them occurs via a set of donor-acceptor hydrogen bond

pairs between the enzyme and its target. Arginine-guanine pairs, with two hydrogen bonds between

the guanidino group of the arginine and the Hoogsteen edge of the purine, are a classic example of such

interaction. We have observed it for a number of the studied enzymes (MvaI, Ecl18kI, ThaI, DpnI)

(Kaus-Drobek, Czapinska et al., 2007; Bochtler et al., 2006; Firczuk, Wojciechowski, Czapinska, Bochtler,

2011; Mierzejewska, Siwek, Czapinska et al., 2014).

We have described a variety of base recognition schemes such as the side chain carboxyl or amide group

bridging of two adjacent bases (Ecl18kI, PspGI, Hpy99I)(Bochtler et al., 2006; Szczepanowski, Carpenter,

Czapinska et al., 2008; Sokolowska, Czapinska, Bochtler, 2009), or a histidine residue hydrogen bonding

(with its main chain and side chain) with two bases of a pair (MvaI)(Kaus-Drobek, Czapinska et al., 2007).

In some cases, an ambiguous hydrogen bonding scheme can lead to degenerate base pair recognition.

This is particularly useful for pseudopalindromic target sequences in which the central base pair must

be accepted in both orientations (e.g. C:G and G:C). We have described such recognition mechanism

in the case of BcnI restriction endonuclease where two histidines can either donate or accept hydrogen

bonds depending on the orientation of the central C:G pair of its target (Sokolowska, Kaus-Drobek,

Czapinska et al., 2007).

Shape complementarity

The second way to discriminate between the target and random sequences makes use of the van der

Waals repulsion. Steric hindrance can be exploited to discriminate between different bases, in particular

thymine and cytosine, but to some extent also between other base combinations. It is one of the means

to distinguish between the C:G|G:C and A:T|T:A pairs at the center of the pseudopalindromic target

sequences. The C:G|G:C pairs have an additional amino group in the central minor groove and thus

should not fit in the binding site shaped to accept the A:T|T:A pairs as observed for example in the MvaI

and Hpy99I endonucleases (Kaus-Drobek, Czapinska et al., 2007; Sokolowska, Czapinska, Bochtler,

2009).

Van der Waals repulsion is also the most common way in which methylation protects the host genome -

the methyl group simply does not fit into the endonuclease active site and thus the modified target

sequence cannot be bound and hydrolyzed. Among the enzymes that we have studied, MvaI

endonuclease was most documented with respect to its methylation sensitivity (Gromova et al., 1991).

Thus, the structure-function relationship could be most thoroughly presented for this example.

The monomeric enzyme interacting with its target sequence in an asymmetric manner, is in some

positions for steric reasons blocked by the presence of the modification in one DNA strand but can still

cleave the other (Kaus-Drobek, Czapinska et al., 2007).

We have next extended the analysis and estimated the magnitude of the steric clash necessary for

the inhibition of the activity of any restriction endonuclease. For this purpose, we have in silico

introduced methyl groups into the DNA bases in all available structures of endonuclease-DNA complexes.

The methyl groups were classified as three classes - the ones introduced by the cognate

methyltransferases (that have biologically evolved to protect the DNA against endonuclease activity),

the ones introduced artificially (either enzymatically or chemically) that are known to be tolerated by


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the specific enzyme and the non-cognate ones that are known to prevent cleavage. The statistical

analysis of the first two classes indicated that the protective and tolerated methyl groups can

be distinguished based on the magnitude of the clashes with approximately 90% sensitivity and

specificity. The steric conflict was then used to predict whether the methyl groups of the third class

would be protective. The analysis indicated that approximately 70% of the effects of artificially

introduced methyl groups were predicted correctly (Mierzejewska, Bochtler, Czapinska, 2016).

Van der Waals attractive forces

Attractive van der Waals forces lie on the interface between direct and indirect sequence recognition.

On one hand they are used as a form of the shape selection. In this role they are most often observed

in modification dependent endonucleases. This group of enzymes is faced with the difficult task to select

for the presence of methyl groups that can only be detected by such forces. This goal is much more

challenging than the specificity towards a lack of modification, that can easily be achieved through strong

repulsive interactions described above. The mechanisms of modification and in particular methyl group

recognition remain controversial. The one most commonly observed by us relies on the interaction of

the group with a face of an aromatic or a π-electron system. We have described such interaction in the

case of 6-methyladenine binding DpnI (Mierzejewska, Siwek, Czapinska et al., 2014) and 5-(hydroxy)

methylcytosine specific EcoKMcrA endonuclease (Czapinska et al., 2018).

Alternatively, attractive van der Waals forces can be used for indirect probing of the base pair steps.

Some consecutive base pairs are held together by stronger stacking interactions than others (i.e. the

overlap between them is larger). The enzymes may indirectly detect their target sequences by probing

the properties of the base pair steps.

DNA bending

There are several approaches used by the RM systems to indirectly detect their targets by probing

the tolerance of particular DNA sequences to deformations. The less severe of them exploit the tendency

of particular DNA sequence to bend or undergo distortions. In our studies the most pronounced use

of such mechanism was observed for DpnI endonuclease. The enzyme detects its target sequence thanks

to a minor deformation driven by the presence of two 6-methyladenine methyl groups in close proximity

in the central major groove (Mierzejewska, Siwek, Czapinska et al., 2014).

Intercalation

In a more extreme scenario, the enzymes intercalate a residue or residues into the DNA stack which

is more feasible for the purine-pyrimidine sequence than for the other base combinations. We have

described such mechanism in a endonuclease and a methyltransferase. In the case of ThaI restriction

endonuclease-DNA complex, each protomer of the dimeric enzyme introduces a methionine residue

in between the last two bases of one strand of its target DNA. This results in the tilt of two central base

pairs of the recognized DNA fragment (Firczuk, Wojciechowski, Czapinska, Bochtler, 2011). In the case

of the M.MpeI methyltransferase two distinct intercalation events were observed that serve two

different purposes. As it is usually observed in this group of enzymes - a glutamine residue is inserted

into the DNA stack in the space created by the flipping of a cytosine into the catalytic pocket of

the enzyme. In M.MpeI-DNA complex, a phenylalanine residue is additionally intercalated between


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the bases of the second DNA strand, which serves the purpose of target sequence recognition

(Wojciechowski, Czapinska, Bochtler, 2013). In both ThaI and M.MpeI, the intercalation is used to detect

the CG target sequence. Interestingly, analogous mechanism of CG sequence recognition was also

described for the eukaryotic Dnmt1 methyltransferase (Song et al., 2011).

Nucleotide flipping

The most radical way of probing the forces holding a DNA helix together relies on flipping of one or both

bases out of the base pair stack. In this way not only the stacking forces are probed but also the number

of hydrogen bonds that keep the two bases together. Extrusion of a base or bases out of the DNA helix

is often combined with the further confirmation of the identity of the flipped base(s) in a binding

pocket(s) of the enzyme. We have observed four applications of the base the flipping phenomenon.

It could serve to skip a single base pair from readout and thus convert a difficult pseudopalindromic

sequence (with a variable pair in the center) into a palindromic one, as observed for the Ecl18kI

endonuclease (Bochtler et al., 2006). It may be applied to verify the number of hydrogen bonds holding

a base pair together to distinguish strong (G:C|C:G) and weak (A:T|T:A) base pars as described by us for

PspGI enzyme (Szczepanowski, Carpenter, Czapinska et al., 2008). It may also be used to detect

the modification of a particular base, which is more difficult when this base is Watson-Crick paired with

its partner as observed for the SRA domains present in the PvuRts1I and TagI enzymes (Kazrani,

Kowalska, Czapinska, Bochtler, 2014; Kisiala, Copelas, Czapinska et al., 2018). Finally, it is necessary

for methyltransferases to gain access to the base that is to be modified as observed for M.MpeI

(Wojciechowski, Czapinska, Bochtler, 2013).

Impact

Our contribution to the understanding of protein-DNA interactions is very useful for the purposes of the

specificity prediction for the proteins of unknown structure and for the modelling of their interactions

with the target DNA. The knowledge obtained through the study was very helpful for the prediction

of the way in which TALE domains interact with their target sequences (decryption of the TALE-code)

(Bochtler, 2012). Our studies of the magnitude of steric conflict necessary to preclude protein-DNA

binding might find use in the prediction of the interaction of transcription factor DNA biding domains

with DNA in eukaryotic organisms.

We have substantially contributed to the understanding of the specificity mechanisms of modification

dependent restriction endonucleases (MDREs). We were among the first groups that structurally

characterized an enzyme specific for 5-hydroxymethylcytosine containing DNA (PvuRts1I endonuclease

of the SRA-PD(D/E)XK type)(Kazrani, Kowalska, Czapinska, Bochtler, 2014). We have determined the first

structures of enzymes of the SRA-HNH (TagI), NEco-HNH (EcoKMcrA) and PD(D/E)XK-wH (DpnI) classes of

REases (Kisiala, Copelas, Czapinska et al., 2018; Czapinska et al., 2018; Siwek, Czapinska et al., 2012).

Our studies of DpnI provided the first structural description of 6-methyladenine recognition by

a restriction endonuclease and one of very few available structures of the protein bound to DNA carrying

this modification (Siwek, Czapinska et al., 2012; Mierzejewska, Siwek, Czapinska et al., 2014).

The structures of the enzymes that we have determined may also have biotechnological applications.

Engineering a strict specificity towards hemimethylated CG sequences into the M.MpeI enzyme would


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provide an opportunity to use it to amplify the DNA while preserving its epigenetic status. PvuRts1I

group of enzymes already finds application in the detection of 5-hydroxymethylcytosine (Wang et al.,

2011; Sun et al., 2013). The structure of the enzyme might guide the improvement of its properties

in terms of stability and selectivity.

Review of the results obtained in presented publications:

1. Czapinska H, Siwek W, Szczepanowski RH, Bujnicki JM, Bochtler M, Skowronek KJ (2019) Crystal

structure and directed evolution of specificity of NlaIV restriction endonuclease. J Mol Biol

doi: 10.1016/j.jmb.2019.04.010.

The manuscript describes one of a few cases of a successful alterations of the endonuclease specificity.

The obtained crystal structure together with the detailed biochemical and bioinformatics analysis

provides clues about the ways of effective engineering of this group of enzymes. The substitutions

located within immediate vicinity of the catalytic apparatus did not bring the desired effects whereas the

ones positioned further away and altering the dimerization mode and/or the peripheral parts of the DNA

binding tunnel proved successful. According to the reported results it should be much easier to engineer

the specificity into the base pairs located at a distance from the active site.

2. Kisiala M, Copelas A, Czapinska H, Xu SY, Bochtler M (2018) Crystal structure of the modification-


The work presented in the manuscript comprises detailed analysis of TagI enzyme, the first structurally

characterized member of the SRA-HNH group of endonucleases. The enzyme uses the SRA domains to

flip and specifically bind the modified cytosine bases of its target sequence and the HNH nuclease dimer

to cleave within the vicinity of the modification site. Interestingly, the structural and biochemical data

suggest that in some sequence contexts the two SRA domains can bind closely located modified cytosine

bases (in fully methylated DNA) and in some it would cause a steric clash between them. This feature

matches the behavior of some eukaryotic SUVH5 proteins which also bind their fully methylated 5mCpG

targets with two SRA domains.

3. Czapinska H, Kowalska M, Zagorskaite E, Manakova E, Slyvka A, Xu SY, Siksnys V, Sasnauskas G,


The publication presents the first biochemical demonstration of weak modification dependent

endonuclease activity of EcoKMcrA. The structure of the enzyme reveals a dimer of two domain

protomers. Its architecture includes a catalytic module composed of two HNH domains (similar to the

one of TagI enzyme) and two modification binding domains of novel fold and DNA binding mode.

The relatively flexible linker between the two domains is likely to be responsible for the broad range

of distances between the modification and cleavage sites.


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4. Mierzejewska K, Bochtler M$, Czapinska H$ (2016) On the role of steric clashes in methylation

control of restriction endonuclease activity. Nucleic Acids Res 44, 485-95.

The manuscript comprises systematic analysis of the magnitude of steric conflict that is significant

enough to have biological implications. We have used the restriction-modification model system that is

ideal to distinguish between clashes that are tolerated and do not affect protein-DNA interaction from

those that prevent DNA cleavage. The RM systems are designed for this purpose, because the inhibition

of the endonuclease activity, resulting from the presence of the methyl groups introduced by the

methyltransferase, is crucial for their proper functioning (disintegration of the foreign DNA and keeping

the host DNA intact). Moreover, we could make use of the thorough information on the effects of in vitro

introduced methyl groups (either chemically or enzymatically) on the activity of various restriction

enzymes freely available in the REBASE database (rebase.neb.com). Finally, thanks to the efforts of our,

and other groups (my contribution to the field is a subject of this application), structural data

are currently available for more than 30 different endonuclease-DNA complexes. The experimentally

determined structures together with the knowledge about the conformation of the methylated bases,

enable relatively accurate prediction of the methyl group positions. We have introduced the methyl

groups in silico into the available endonuclease-DNA complex structures and quantified the steric clashes

that would occur if the DNA was methylated. We have next correlated the steric conflicts with the

biological effect of the methylations (their protective effect against the endonuclease cleavage or lack

thereof). Our analysis determined the van der Waals radii overlap larger than 1.1 Å and the volume

overlap larger than 48 Å3 as the best indicators of the protective character of an introduced methyl

group, i.e. perturbation of the protein-DNA interaction (0.1 Å was deducted from each interatomic

distance to allow for coordinate errors and adaptive fit).

5. Kazrani AA, Kowalska M, Czapinska H, Bochtler M (2014) Crystal structure of the 5hmC specific

endonuclease PvuRts1I. Nucleic Acids Res 42, 5929-36

The work presented in the paper was aimed at a structural characterization of the

5-hydroxymethylcytosine (and glucosyl-5-hydroxymethylcytosine) specific restriction endonuclease

PvuRts1I from Proteus vulgaris. This was the first published structure of an enzyme with such selectivity.

The analysis of the structure revealed that the enzyme is built of two domains:

an unspecific endonuclease domain of a PD-(D/E)XK type and a target recognition domain of an SRA

(SET and RING finger associated) fold. The catalytic type of PvuRts1I has been bioinformatically predicted

before (Bujnicki and Rychlewski, 2001) but the description of the DNA binding domain was to our

knowledge truly novel. The SRA domains are known to recognize modified bases while flipping them out

of the DNA stack and binding in dedicated pockets on their surface. Therefore, even in an absence of

a structural proof of this concept we predicted that PvuRts1I should act accordingly and pinpointed the

putative flipped base binding pocket and some of the key residues responsible for target recognition.

Since the enzyme is known to be a dimer, we further predicted that it will work in a FokI like manner

and presented the model of the putative PvuRts1I-DNA complex. Our model predicted that two

unspecific endonuclease domains would dimerize on the DNA half way between of the two target sites

recognized by the SRA domains and was later structurally confirmed by others (Horton et al., 2014).


13

6. Mierzejewska K*, Siwek W*, Czapinska H*, Kaus-Drobek M, Radlinska M, Skowronek K, Bujnicki JM,


Res 42, 8745-54.

The aim of this work was to analyze the mechanistic basis of the 6-methyladenine specificity of the DpnI

catalytic domain and the mode of its binding to the G6mATC target sequence. We have determined

the structure of the tertiary complex of DpnI and two DNA molecules bound to its two domains. It led to

the observation that the two methyl groups of the symmetry related 6mA residues in two DNA strands of

DpnI target are located in very close proximity and bound in a single hydrophobic pocket of the enzyme.

The pocket is formed by a long β-hairpin that wedges into the DNA major groove. The key residues

involved in the pocket formation include a tryptophan that thanks to its bulky side chain stacks against

both methyl groups at a time.

We have searched the Protein Data Bank for all structures that contain DNA fragments of nonmethylated

GATC sequence. The analysis of the selected structures revealed that in 80% of the cases the N6 atoms

of the adenines are so close that the introduction of the two methyl groups would have to be followed

by an adjustment of the DNA conformation. Thus, we concluded that DpnI-DNA binding is in part driven

by the fact that its target is pre-deformed already prior to the interaction with the protein.

7. Wojciechowski M, Czapinska H, Bochtler M (2013) CpG underrepresentation and the bacterial

CpG-specific DNA methyltransferase M.MpeI. Proc Natl Acad Sci USA 110, 105-10.

This work was initiated by the search for a prokaryotic counterpart of the eukaryotic CpG

methyltransferases (Dnmts). The Dnmt proteins are large molecules of a very complex architecture and

the structure of none of them was known at a time. We have followed a standard approach used

in structural biology to find a simpler prokaryotic system that would be easier to work with and at the

same time might help to elucidate the specificity mechanism of its complicated eukaryotic counterparts.

This strategy led to the finding of Mycoplasma penetrans M.MpeORF4940P as a likely candidate. After

proving the activity of the protein as a C5-cytosine methyltransferase, we have termed the enzyme

M.MpeI according to the nomenclature guidelines.

The structure of M.MpeI in complex with DNA has revealed that the enzyme has the expected

S-adenosylmethionine dependent methyltransferase fold and flips its target cytosine to introduce

the modification. However, it has also shown an unexpected feature of the protein, namely, that it

intercalates a phenylalanine residue in between the two bases of the second strand of its target.

The CpG (pyrimidine-purine) step is easier to unstack than the other base combination and thus favored

by the enzyme. In this way M.MpeI enhances the fidelity of the recognition of its short CpG target

sequence via indirect interactions. Once the Dnmt1 methyltransferase structure has been elucidated

it became clear that the same mechanism is also applied by the eukaryotic enzyme.


14

8. Siwek W, Czapinska H, Bochtler M, Bujnicki JM, Skowronek K (2012) Crystal structure and

mechanism of action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI.

Nucleic Acids Res 40, 7563-72.

This publication presents the work that aimed at the structural characterization of G6mATC specific DpnI

restriction endonuclease. We have shown that the enzyme is a two domain protein with a catalytic

domain of a PD-(D/E)XK fold and an auxiliary winged helix domain. We have further shown that each

of the domains displayed selectivity for both the DNA sequence and its methylation status. Thus, DpnI

has been classified as both type IIM (modification specific) as well as type IIE (with the activity enhanced

by the presence of an additional target site) restriction endonuclease. The winged helix domain (similarly

as the catalytic domain presented above) recognizes both methyl groups in a single hydrophobic cavity

that is in this case formed by the residues of one face of an α-helix that wedges into the major groove

of the target DNA. In the presented structure the catalytic domain of the enzyme was in a DNA unbound

form therefore we have continued the studies and determined the structure of a tertiary complex

described above (Mierzejewska et al., 2014).

9. Firczuk M, Wojciechowski M, Czapinska H, Bochtler M (2011) DNA intercalation without flipping in

the specific ThaI-DNA complex. Nucleic Acids Res 39, 744-54.

The studies of ThaI enzyme were launched as a result of a search for a CpG specific endonuclease. A pair

of two enzymes (cytosine C5 methylation sensitive HpaII and insensitive MspI) is currently used to detect

the epigenetic status of DNA, via generation and analysis of two restriction patterns, that depend on

the methylation state of the studied material (Yaish et al., 2014; Oda et al., 2009; Zilberman and

Henikoff, 2007; Takamiya et al., 2006). The two enzymes are however both specific for the CCGG target,

and do not detect the modifications of CpG dinucleotide in other sequence contexts. ThaI endonuclease

(with CGCG target site) belonged to a set of enzymes that we have selected as potential candidates

for engineering of an endonuclease with a specificity reduced to the CG sequence only. We have not

tried the commercially used pair of endonucleases for patent reasons. The engineering effort was

abandoned as soon as it was clear that the enzyme acts as a dimer and thus it will be hard to get rid of

its selectivity for one half of the symmetric target sequence only. Nonetheless the enzyme proved

to be interesting from a structural perspective. It detects its target sequence via intercalation of two

methionine residues between the C and G bases of a weak pyrimidine-purine DNA step. This feature

proved even more interesting once it became clear that the same mechanism is observed with

the CpG specific M.MpeI and Dnmt1 methyltransferases.

10. Sokolowska M, Czapinska H, Bochtler M (2011) Hpy188I-DNA pre- and post-cleavage complexes -


This and the following set of papers originated from two observations: (i) that the stagger between

the scissile bonds in the two DNA strands determines the relative positions of the two active sites

of a (dimeric) endonuclease that is supposed to cleave them; and (ii) that most of the restriction

endonucleases structurally characterized at a time cleave DNA to produce either products with blunt

ends or 4 nucleotide overhangs. We have concluded that the restriction endonucleases of a novel fold


15

or unusual properties are more likely to be found among the “exotic” stagger producing ones. Therefore,

we have systematically structurally characterized a set of such enzymes.

The Hpy188I restriction endonuclease cleaves DNA leaving products with single nucleotide 3′-overhangs.

We have determined the structure of the enzyme, which revealed a fold of a GIY-YIG type endonuclease

built around a conserved antiparallel β-sheet. This fold was not previously observed in any structurally

characterized restriction endonuclease. The active site of the enzyme contains a LVY and KIG motifs

instead of the canonical logo. Our structures of the enzyme-DNA complex in both pre- and post-reaction

state made it possible to pinpoint the first and not the second tyrosine residue of the canonical motif

(Y and not K in Hpy188I) as the general base. This is in contrast to what was expected before our work

in the absence of any GIY-YIG nuclease-DNA complex structure. Our studies also suggest that the

tyrosine should occur in its phenolate form to perform its catalytic function. Finally, the active site of the

enzyme was shown to contain a single metal ion that is thought to stabilize the leaving group 3′ oxygen.

11. Sokolowska M, Czapinska H, Bochtler M (2009) Crystal structure of the ββα-Me restriction

endonuclease Hpy99I. Nucleic Acids Res 37, 3799-810.

The Hpy99I restriction endonuclease was selected for our studies because it cleaves DNA with a five

nucleotide 5’ cleavage stagger. The structure of the enzyme has proved it to be a dimer, with each of the

protomers built of two repeats and an additional β-barrel domain. The repeats have a zinc finger type

fold that coordinates a zinc ion via four cysteine residues. One of the repeats in each protomer hosts the

active site of the enzyme of an HNH type and the other has a DNA binding role. This was the first

structural description of a restriction nuclease of this catalytic type. The structures of Hpy99I

and Hpy188I, described above, led to the determination of a few structural analogies between the HNH

and GIY-YIG active sites that were anticipated but lacked experimental evidence (Gasiunas et al., 2008;

Belfort et al., 2005),. Both harbor a single metal ion that serves to stabilize the leaving group 3′ oxygen.

In both the requirements for the metal identity are not strict. The metal ion is coordinated by an acidic

residue that is located in an α-helix. Finally, both types of enzymes use a residue located in a β-hairpin

as a general base, although it is a histidine in the HNH and tyrosine in the GIY-YIG nucleases. The overall

folds of the two classes of enzymes are however substantially different and thus we expect

the similarities to result from convergent evolution. It needs to be noted that the work on both groups of

enzymes was completed independent of and simultaneously with the structural studies of two

endonucleases of the same classes performed by the laboratory of Prof. Barry Stoddard (Mak et al.,

2010; Shen et al., 2010).

12. Szczepanowski RH, Carpenter MA, Czapinska H, Zaremba M, Tamulaitis G, Siksnys V, Bhagwat AS,

Bochtler M (2008) Central base pair flipping and discrimination by PspGI. Nucleic Acids Res 36,

6109-17.

The work on PspGI restriction endonuclease was aimed at the elucidation of this enzyme specificity

for the two central base pairs of its pseudopalindromic target sequence. The dimeric enzyme flips them

out of the DNA stack and binds in dedicated pockets on the surface of its protomers. However in contrast

to its homologs it displays preference for the A:T (and for symmetry reasons also T:A) bases in flipped


16

position. We were interested in a mechanism by which the enzyme built of two identical protomers can

accept in the same pocket two bases as different as A and T and at the same time discriminate against

G and C. The biochemical and structural data point to a dual specificity mechanism. PspGI senses

the properties of the central base pair prior to the flipping event (number of hydrogen bonds that hold

them together and stacking interactions with the neighboring bases) and further verifies their identity

in the binding pockets.

13. Sokolowska M, Kaus-Drobek M, Czapinska H, Tamulaitis G, Siksnys V, Bochtler M (2007) Restriction

endonucleases that resemble a component of the bacterial DNA repair machinery. Cell Mol Life Sci

64, 2351-57.

The paper describes our analysis of the similarities between two restriction endonucleases (BcnI and

MvaI) and MutH enzyme, which belongs to the bacterial DNA repair system. MutH is a nickase and

requires the presence of other components of the system to perform the cleavage reaction whereas

the two nucleases are active in the absence of additional proteins. However, there are striking

similarities between the three enzymes. In contrast to most endonucleases all three are monomeric and

interact with their target sequence asymmetrically. They are all built of two lobes out of which one has

a catalytic role and the other is involved in target sequence recognition. Finally, all three enzymes have

a hinge region between the two lobes which allows them to open up and release the reaction product.

This is crucial for the endonucleases which have to then bind the target DNA in reverse orientation

to perform cleavage of the second DNA strand.

14. Sokolowska M, Kaus-Drobek M, Czapinska H, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V,



The rationale for the studies of BcnI restriction endonuclease came from its unusual cleavage stagger

that gives rise to DNA fragments with one nucleotide 5′ overhang. Against expectations the structure

of the enzyme has proved it to be a monomer and to form a distinct set of interactions with each strand

of its target sequence. From the monomeric character of the enzyme we have deduced that it requires

two sequential DNA binding events to introduce double strand DNA break. Finally, we have also

determined the structure of the enzyme with the central base pair bound in reversed orientation, which

provided hints about the recognition of this pseudopalindromic position of its target sequence. Both C:G

and G:C pairs can be accepted by the enzyme in this position most likely thanks to the switch of the

protonation state of the two histidines on the major groove side. In this way the two residues can either

donate or accept hydrogen bonds from the DNA, depending on its orientation. The A:T and T:A pairs are

discriminated most likely for steric reasons (clash of the thymine exocyclic methyl group with the

protein).


17

15. Kaus-Drobek M, Czapinska H, Sokolowska M, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V,



MvaI enzyme shares the cleavage stagger with BcnI endonuclease presented above. We have studied the

two enzymes due to their different specificities for the central pseudopalindromic pair of the target

sequence (T:A|A:T for MvaI and C:G|G:C for BcnI) and analogous preferences for the other base pairs

(CCXGG). The structures confirmed the expectation that the outer pairs are to some extent recognized

analogously by the two proteins, although a substantial evolutionary drift can be observed. In the central

position MvaI applies the same principle of steric hindrance to prevent binding of non-cognate C:G|G:C

pairs. However, the clash is in this case expected not on the major as for BcnI but on the minor groove

side, where the exocyclic amino group of guanine is predicted to clash with the protein.

16. Bochtler M, Szczepanowski RH, Tamulaitis G, Grazulis S, Czapinska H, Manakova E, Siksnys V (2006)

Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. EMBO Journal 25,

2219-29.

The crystal structure of Ecl18kI-DNA complex brought a first example of a nucleotide flipping enzyme

among restriction endonucleases. The finding proved even more interesting once it became clear that

the enzyme uses this phenomenon to make a five base pair stagger imitate the four base pair one.

Thus, while preserving the dimerization interface and positions of the active sites with the abundant

group of endonucleases that produce four nucleotide 5’ overhangs, it adapted to a radically different

type of target sequence. Flipping of the bases of the central pseudopalindromic pair further serves the

enzyme to skip them from readout, which is accomplished through binding of these bases in relatively

unspecific pockets on the enzyme surface.

References

Belfort M, Stoddard BL, Wood DW, Derbyshire V (2005) Homing Endonucleases and Inteins. Springer,

Berlin.

Bochtler M (2012) Structural basis of the TAL effector-DNA interaction. Biol Chem 393:1055-66.

Bochtler M, Szczepanowski RH, Tamulaitis G, Grazulis S, Czapinska H, Manakova E, Siksnys V (2006)

Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. EMBO Journal

25, 2219-29.

Bujnicki JM, Rychlewski L (2001) Grouping together highly diverged PD-(D/E)XK nucleases and

identification of novel superfamily members using structure-guided alignment of sequence profiles.

J Mol Microbiol Biotechnol 3, 69–72.

Chandrasegaran S, Carroll D (2016) Origins of Programmable Nucleases for Genome Engineering. J Mol

Biol 428:963-89.

Chen L, MacMillan AM, Verdine GL (1993) Mutational separation of DNA binding from catalysis in a DNA

cytosine methyltransferase. J Am Chem Soc 115:5318-19.


18

Cheng X, Kumar S, Posfai J, Pflugrath JW, Roberts RJ (1993) Crystal structure of the HhaI DNA

methyltransferase complexed with S-adenosyl-L-methionine. Cell 74:299-307.

Cheng X (1995) Structure and function of DNA methyltransferases. Annu Rev Biophys Biomol Struct

24:293-318.

Cheng X, Roberts RJ (2001) AdoMet-dependent methylation, DNA methyltransferases and base flipping.

Nucleic Acids Res 29:3784-95.

Chevalier B, Monnat RJ Jr, Stoddard BL (2005) The LAGLIDADG Homing Endonuclease Family (Homing

Endonucleases and Inteins) Springer, Berlin.

Czapinska H, Kowalska M, Zagorskaite E, Manakova E, Slyvka A, Xu SY, Siksnys V, Sasnauskas G,


Czapinska H, Siwek W, Szczepanowski RH, Bujnicki JM, Bochtler M, Skowronek KJ (2019) Crystal structure

and directed evolution of specificity of NlaIV restriction endonuclease. J Mol Biol

doi: 10.1016/j.jmb.2019.04.010.

Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A, Keren M, Amitai G, Sorek R (2018) Systematic

discovery of antiphage defense systems in the microbial pangenome. Science 359:6379 (eaar4120).

Firczuk M, Wojciechowski M, Czapinska H, Bochtler M (2011) DNA intercalation without flipping in the

specific ThaI-DNA complex. Nucleic Acids Res 39, 744-54.

Gabbara S, Sheluho D, Bhagwat AS (1995) Cytosine methyltransferase from Escherichia coli in

which active site cysteine is replaced with serine ispartially active. Biochemistry 34:8914-23.

Gasiunas G, Sasnauskas G, Tamulaitis G, Urbanke C, Razaniene D, Siksnys V (2008) Tetrameric restriction

enzymes: expansion to the GIY-YIG nuclease family. Nucleic Acids Res 36:938-949.

Gaur V, Wyatt HDM, Komorowska W, Szczepanowski RH, de Sanctis D, Gorecka KM, West SC,

Nowotny M (2015) Structural and Mechanistic Analysis of the Slx1-Slx4 Endonuclease. Cell Rep

10:1467-1476.

Gnyszka A, Jastrzebski Z, Flis S (2013) DNA methyltransferase inhibitors and their emerging role in

epigenetic therapy of cancer. Anticancer Res 33:2989-96.

Gromova ES, Kubareva EA, Vinogradova MN, Oretskaya TS, Shabarova ZA (1991) Peculiarities of

recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII. J Mol

Recognit 4, 133–141.

Gromova ES, Khoroshaev AV (2003) Prokaryotic DNA methyltransferases: the structure and the

mechanism of interaction with DNA. Mol Biol (Mosk) 37:300-14.

Horton JR, Borgaro JG, Griggs RM, Quimby A, Guan S, Zhang X, Wilson GG, Zheng Y, Zhu Z, Cheng X

(2014) Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with

DNA. Nucleic Acids Res 42:7947-7959.

Horton NC, Newberry KJ, Perona JJ (1998) Metal ion-mediated substrate-assisted catalysis in type II

restriction endonucleases. Proc Natl Acad Sci USA 95:13489-94.

Horton NC, Perona JJ (2004) DNA cleavage by EcoRV endonuclease: two metal ions in three metal ion

binding sites. Biochemistry 43:6841-57.


19

Kaus-Drobek M, Czapinska H, Sokolowska M, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V,



Kazrani AA, Kowalska M, Czapinska H, Bochtler M (2014) Crystal structure of the 5hmC specific

endonuclease PvuRts1I. Nucleic Acids Res 42, 5929-36.

Kim YC, Grable JC, Love R, Greene PJ, Rosenberg JM (1990) Refinement of EcoRI endonuclease crystal

structure: a revised protein chain tracing. Science 249:1307-9.

Kisiala M, Copelas A, Czapinska H, Xu SY, Bochtler M (2018) Crystal structure of the modification-


Klimasauskas S, Kumar S, Roberts RJ, Cheng X (1994) HhaI methyltransferase flips its target base out of

the DNA helix. Cell 76:357-69.

Liu H, Naismith JH (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site

plasmid mutagenesis protocol. BMC Biotechnol 8:91.

Loenen WA, Raleigh EA (2014) The other face of restriction: modification-dependent enzymes. Nucleic

Acids Res 42:56-69.

Lyko F (2018) The DNA methyltransferase family: a versatile toolkit for epigenetic regulation. Nat Rev

Genet. 19:81-92.

Mak AN, Lambert AR, Stoddard BL (2010) Folding, DNA recognition, and function of GIY-YIG

endonucleases: crystal structures of R.Eco29kI. Structure 18:1321-31.

McClarin JA, Frederick CA, Wang BC, Greene P, Boyer HW, Grable J, Rosenberg JM (1986) Structure of the

DNA-Eco RI endonuclease recognition complex at 3 A resolution. Science 234:1526-41.

Mierzejewska K, Bochtler M, Czapinska H (2016) On the role of steric clashes in methylation control of

restriction endonuclease activity. Nucleic Acids Res 44, 485-95.

Mierzejewska K, Siwek W, Czapinska H, Kaus-Drobek M, Radlinska M, Skowronek K, Bujnicki JM,


Res 42, 8745-54.

Oda M, Glass JL, Thompson RF, Mo Y, Olivier EN, Figueroa ME, Selzer RR, Richmond TA, Zhang X,

Dannenberg L, Green RD, Melnick A, Hatchwell E, Bouhassira EE, Verma A, Suzuki M, Greally JM

(2009) High-resolution genome-wide cytosine methylation profiling with simultaneous copy number

analysis and optimization for limited cell numbers. Nucleic Acids Res 37:3829-39.

Orlowski J, Bujnicki JM (2008) Structural and evolutionary classification of Type II restriction enzymes

based on theoretical and experimental analyses. Nucleic Acids Res 36:3552-69.

Pechalrieu D, Etievant C, Arimondo PB (2017) DNA methyltransferase inhibitors in cancer: From

pharmacology to translational studies. Biochem Pharmacol 1;129:1-13.

Pingoud A, Fuxreiter M, Pingoud V, Wende W (2005) Type II restriction endonucleases: structure and

mechanism. Cell Mol Life Sci 62:685-707.

Reinisch KM, Chen L, Verdine GL, Lipscomb WN (1995) The crystal structure of HaeIII methyltransferase

convalently complexed to DNA: an extrahelical cytosine and rearranged base pairing. Cell 82:143-53.


20

Roberts RJ (2005) How restriction enzymes became the workhorses of molecular biology. Proc Natl Acad

Sci USA 102:5905-8.

Saldívar-González FI, Gómez-García A, Chávez-Ponce de León DE, Sánchez-Cruz N, Ruiz-Rios J, Pilón-

Jiménez BA, Medina-Franco JL (2018) Inhibitors of DNA Methyltransferases From Natural Sources: A

Computational Perspective. Front Pharmacol 9:1144.

Shao C, Wang C, Zang J (2014) Structural basis for the substrate selectivity of PvuRts1I, a 5-

hydroxymethylcytosine DNA restriction endonuclease Acta Crystallogr Sect D 70:2477-2486.

Shen BW, Heiter DF, Chan SH, Wang H, Xu SY, Morgan RD, Wilson GG, Stoddard BL.(2010) Unusual target

site disruption by the rare-cutting HNH restriction endonuclease PacI. Structure 18:734-43.

Shen JC, Rideout WM, 3rd, Jones PA (1992) High frequency mutagenesis by a DNA methyltransferase.

Cell 71:1073–1080.

Shen JC, Rideout WM, 3rd, Jones PA (1994) The rate of hydrolytic deamination of 5-methylcytosine in

double-stranded DNA. Nucleic Acids Res 22:972–976.

Siedlecki P, Zielenkiewicz P (2006) Mammalian DNA methyltransferases. Acta Biochim Pol 53:245-56.

Siwek W, Czapinska H, Bochtler M, Bujnicki JM, Skowronek K (2012) Crystal structure and mechanism of

action of the N6-methyladenine-dependent type IIM restriction endonuclease R.DpnI. Nucleic Acids

Res 40, 7563-72.

Sokolowska M, Czapinska H, Bochtler M (2011) Hpy188I-DNA pre- and post-cleavage complexes -


Sokolowska M, Czapinska H, Bochtler M (2009) Crystal structure of the ββα-Me restriction endonuclease

Hpy99I. Nucleic Acids Res 37, 3799-810.

Sokolowska M, Kaus-Drobek M, Czapinska H, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V,



Song J, Rechkoblit O, Bestor TH, Patel DJ (2011) Structure of DNMT1-DNA complex reveals a role for

autoinhibition in maintenance DNA methylation. Science 25:1036-40.

Sun Z, Terragni J, Borgaro JG, Liu Y, Yu L, Guan S, Wang H, Sun D, Cheng X, Zhu Z, Pradhan S, Zheng Y

(2013) High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse

embryonic stem cells. Cell Rep 3:567-76.

Szczepanowski RH, Carpenter MA, Czapinska H, Zaremba M, Tamulaitis G, Siksnys V, Bhagwat AS,

Bochtler M (2008) Central base pair flipping and discrimination by PspGI. Nucleic Acids Res 36, 6109

Takamiya T, Hosobuchi S, Asai K, Nakamura E, Tomioka K, Kawase M, Kakutani T, Paterson AH,

Murakami Y, Okuizumi H (2006) Restriction landmark genome scanning method using isoschizomers

(MspI/HpaII) for DNA methylation analysis. Electrophoresis 27:2846-56.

Wang H, Guan S, Quimby A, Cohen-Karni D, Pradhan S, Wilson G, Roberts RJ, Zhu Z, Zheng Y (2011)

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in

mapping genomic 5-hydroxymethylcytosine. Nucleic Acids Res 39:9294-305.


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Winkler FK, Banner DW, Oefner C, Tsernoglou D, Brown RS, Heathman SP, Bryan RK, Martin PD,

Petratos K, Wilson KS (1993) The crystal structure of EcoRV endonuclease and of its complexes with

cognate and non-cognate DNA fragments. EMBO J 12:1781-95.

Wojciechowski M, Czapinska H, Bochtler M (2013) CpG underrepresentation and the bacterial CpG-

specific DNA methyltransferase M.MpeI. Proc Natl Acad Sci USA 110, 105-10.

Yaish MW, Peng M, Rothstein SJ (2014) Global DNA methylation analysis using methyl-sensitive

amplification polymorphism (MSAP). Methods Mol Biol 1062:285-98.

Zilberman D, Henikoff S (2007) Genome-wide analysis of DNA methylation patterns. Development.

134:3959-65.

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