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DNA methylation is an important epigenetic mechanism essential for transposon silencing and genome integrity. Across evolution, the substrates of DNA methylation have diversified between kingdoms. In plants, chromomethylase3 (CMT3) and CMT2 mediate CHG and CHH methylation, respectively. However, how these two methyltransferases diverge on substrate specificities during evolution remains unknown. Here, we reveal that CMT2 originates from a duplication of an evolutionarily ancient CMT3 in flowering plants. Lacking a key arginine residue recognizing CHG in CMT2 impairs its CHG methylation activity in most flowering plants. An engineered V1200R mutation empowers CMT2 to restore CHG and CHH methylations in Arabidopsis cmt2cmt3 mutant, testifying a loss-of-function effect for CMT2 during evolution. CMT2 has evolved a long and unstructured amino terminus critical for protein stability, especially under heat stress, and is plastic to tolerate various natural mutations. Together, this study reveals the mechanism of chromomethylase divergence for context-specific DNA methylation in plants and sheds important lights on DNA methylation evolution and function.

Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Herein, a novel label-free photoelectrochemical (PEC) sensing platform with near-zero background noise was developed for M.SssI CpG methyltransferase (M.SssI MTase) activity assay based on a new Schottky junction of Bi 2 S 3 /Ti 3 C 2 nanosheets. The proposed PEC sensor exhibited a low detection limit and a high signal-to-noise ratio for M.SssI MTase assay.

DNA methylation catalyzed by M.SssI methyltransferases (MTase) has important roles in gene expression and other cellular activities, and relates to some diseases, especially cancers. Therefore, it is important to develop a sensitive sensing platform for M.SssI MTase activity assay. Here, taking zeolitic imidazolate framework-8 (ZIF-8) polyhedra as the carriers of graphene quantum dots (GQDs), GQDs-embedded ZIF-8 polyhedra (denoted as GQDs@ZIF-8 polyhedra) were successfully prepared and used as the multi-functional signal quencher to construct a novel signal-off photoelectrochemical (PEC) biosensor for M.SssI MTase activity assay. Firstly, the indium tin oxide (ITO) slice was modified with TiO 2 , poly(diallyldimethylammonium chloride) and CdTe quantum dots (QDs). The obtained electrode was used as the photoelectrode and labeled as ITO/TiO 2 /CdTe QDs. Then, single-stranded DNA (S1) was anchored on the photoelectrode surface via S-Cd bond. After hybridization between S1 and biotinylated single-stranded DNA (S2), the streptavidin (SA)-labeled GQDs@ZIF-8 polyhedra were introduced to the modified electrode via the specific reaction between biotin and SA. As the signal quencher, GQDs@ZIF-8 polyhedra could not only inhibit the photocurrent signal of the ITO/TiO 2 /CdTe QDs electrode due to the steric hindrance effect, but also act as peroxidase mimetics to catalyze precipitation reaction of 4-chloro-1-naphthol, resulting in the evident depression of the photocurrent signal. For the specially designed S1/S2 double-strand DNA, the decreased photocurrent was quantitatively correlated with the M.SssI MTase activity (linear response range, 0.005-150 U mL -1 ; detection limit, 0.004 U mL -1 ). The developed GQDs@ZIF-8 polyhedra and related PEC biosensor may have potential applications in clinical research and disease diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single-molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. Sss I MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. Sss I, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently, the streptavidin-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. Sss I concentration in the concentration range of 0.001-1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. Sss I in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. Sss I inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Methylation of cytosine is the common epigenetic modification in genomes ranging from bacteria to mammals, and aberrant methylation leads to human diseases including cancer. Recognition of a cognate DNA sequence by DNA methyltransferases and flipping of a target base into the enzyme active site pocket are the key steps in DNA methylation. Using molecular dynamics simulations and enhanced sampling techniques here we elucidate the role of conformational fluctuations of protein and active or passive involvement of protein elements that mediate base flipping and formation of the closed catalytic complex. The free energy profiles for the flipping of target cytosine into the enzyme active site support the major groove base eversion pathway; and the results show that the closed state of enzyme increases the free energy barrier, whereas the open state reduces it. We found that the interactions of the key loop residues of protein with cognate DNA altered the protein motions, and modulation of protein fluctuations relates to the closed catalytic complex formation. Methylation of cytosine in the active site of the closed complex destabilizes the interactions of catalytic loop residues with cognate DNA and reduces the stability of the closed state. Our study provides microscopic insights on the base flipping mechanism coupled with enzyme's loop motions and provides evidence for the role of conformational fluctuations of protein in the enzyme-catalyzed DNA processing mechanism.

Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.

Although the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear because of the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here, we present mirror bisulfite sequencing for 5hmC detection at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semiconservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. The glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand, whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired liver tissues were mapped genomewide.

DNA methyltransferase (MTase) has a crucial role in many biological processes, its abnormal expression level has been regarded as a predictive cancer biomarker. Herein, a sensitive fluorescence method based on thermosensitive poly (N-isopr-opylacrylamide) was developed to assay of M.SssI activity. When the M.SssI was introduced, dsDNA was methylated at palindromic sequence 5'-C m CGG-3' and became resistant to cleavage by the endonuclease HpaII. Therefore, a biotin modified ssDNA and a FAM modified ssDNA were designed including the recognized sites for both methyltransferase M.SssI and endonuclease HpaII. By SA-biotin intereaction, the DNA was conjugated to thermosensitive poly (N-isopropylacrylamide) modified by SA, the methylated substrate fluorescence was increased with the concentration of M.SssI increasing. The proposed method has a low detection limit of 0.18 U/mL. This simple method can be a useful tool to apply in diagnosis and biomedical research, which was successfully investigated in the serum sample., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Type II restriction⁻modification (RM) systems are the most widespread bacterial antiviral defence mechanisms. DNA methyltransferase SsoII (M.SsoII) from a Type II RM system SsoII regulates transcription in its own RM system in addition to the methylation function. DNA with a so-called regulatory site inhibits the M.SsoII methylation activity. Using circular permutation assay, we show that M.SsoII monomer induces DNA bending of 31° at the methylation site and 46° at the regulatory site. In the M.SsoII dimer bound to the regulatory site, both protein subunits make equal contributions to the DNA bending, and both angles are in the same plane. Fluorescence of TAMRA, 2-aminopurine, and Trp was used to monitor conformational dynamics of DNA and M.SsoII under pre-steady-state conditions by stopped-flow technique. Kinetic data indicate that M.SsoII prefers the regulatory site to the methylation site at the step of initial protein⁻DNA complex formation. Nevertheless, in the presence of S -adenosyl-l-methionine, the induced fit is accelerated in the M.SsoII complex with the methylation site, ensuring efficient formation of the catalytically competent complex. The presence of S -adenosyl-l-methionine and large amount of the methylation sites promote efficient DNA methylation by M.SsoII despite the inhibitory effect of the regulatory site.

DNA methylation is a common modification occurring in all living organisms. 5-methylcytosine, which is produced in a reaction catalysed by C5-methyltransferases, can spontaneously undergo deamination to thymine, leading to the formation of T:G mismatches and C→T transitions. In Escherichia coli K-12, such mismatches are corrected by the Very Short Patch (VSP) repair system, with Vsr endonuclease as the key enzyme. Neisseria meningitidis possesses genes that encode DNA methyltransferases, including C5-methyltransferases. We report on the mutagenic potential of the meningococcal C5-methyltransferases M.NmeDI and M.NmeAI resulting from deamination of 5-methylcytosine. N. meningitidis strains also possess genes encoding potential Vsr endonucleases. Phylogenetic analysis of meningococcal Vsr endonucleases indicates that they belong to two phylogenetically distinct groups (type I or type II Vsr endonucleases). N. meningitidis serogroup C (FAM18) is a representative of meningococcal strains that carry two Vsr endonuclease genes (V.Nme18IIP and V.Nme18VIP). The V.Nme18VIP (type II) endonuclease cut DNA containing T:G mismatches in all tested nucleotide contexts. V.Nme18IIP (type I) is not active in vitro, but the change of Tyr69 to His69 in the amino acid sequence of the protein restores its endonucleolytic activity. The presence of tyrosine in position 69 is a characteristic feature of type I meningococcal Vsr proteins, while type II Vsr endonucleases possess His69. In addition to the T:G mismatches, V.Nme18VIP and V.Nme18IIPY69H recognize and digest DNA with T:T or U:G mispairs. Thus, for the first time, we demonstrate that the VSP repair system may have a wider significance and broader substrate specificity than DNA lesions that only result from 5-methylcytosine deamination.

DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity.

The bacterium Deinococcus radiodurans-like all other organisms-introduces nucleotide modifications into its ribosomal RNA. We have previously found that the bacterium contains a Carbon-5 methylation on cytidine 2499 of its 23S ribosomal RNA, which is so far the only modified version of cytidine 2499 reported. Using homology search, we identified the open reading frame DR_0049 as the primary candidate gene for the methyltransferase that modifies cytidine 2499. Mass spectrometric analysis demonstrated that recombinantly expressed DR0049 protein methylates E. coli cytidine 2499 both in vitro and in vivo. We also inactivated the DR_0049 gene in D. radiodurans through insertion of a chloramphenicol resistance cassette. This resulted in complete absence of the cytidine 2499 methylation, which all together demonstrates that DR_0049 encodes the methyltransferase producing m(5)C2499 in D. radiodurans 23S rRNA. Growth experiments disclosed that inactivation of DR_0049 is associated with a severe growth defect, but available ribosome structures show that cytidine 2499 is positioned very similar in D. radiodurans harbouring the modification and E. coli without the modification. Hence there is no obvious structure-based explanation for the requirement for the C2499 posttranscriptional modification in D. radiodurans.

DNA methyltransferases (MTases) catalyze the transfer of the activated methyl group of the cofactor S-adenosyl-l-methionine (AdoMet or SAM) to the exocyclic amino groups of adenine or cytosine or the C5 ring atom of cytosine within specific DNA sequences. The DNA adenine-N6 MTase from Thermus aquaticus (M.TaqI) is also capable of coupling synthetic N-adenosylaziridine cofactor analogues to its target adenine within the double-stranded 5'-TCGA-3' sequence. This M.TaqI-mediated coupling reaction was exploited to sequence-specifically deliver fluorophores and biotin to DNA using N-adenosylaziridine derivatives carrying reporter groups at the 8-position of the adenine ring. However, these 8-modified aziridine cofactors were poor substrates for the DNA cytosine-C5 MTase from Haemophilus haemolyticus (M.HhaI). Based on the crystal structure of M.HhaI in complex with a duplex oligodeoxynucleotide and the cofactor product, we synthesized a stable 7-deazaadenosylaziridine derivative with a biotin group attached to the 7-position via a flexible linker. This 7-modified aziridine cofactor can be efficiently used by M.HhaI for the direct, quantitative and sequence-specific delivery of biotin to the second cytosine within 5'-GCGC-3' sequences in short duplex oligodeoxynucleotides and plasmid DNA. In addition, we demonstrate that biotinylation by M.HhaI depends on the methylation status of the target cytosine and, thus, could provide a method for cytosine-C5 DNA methylation detection in mammalian DNA.

Different mutagenic effects are generated by DNA oxidation that implies the formation of radical cation states (so-called holes) on purine nucleobases. The interaction of DNA with proteins may protect DNA from oxidative damage owing to hole transfer (HT) from the stack to aromatic amino acids. However, how protein binding affects HT dynamics in DNA is still poorly understood. Here, we report a computational study of HT in DNA complexes with methyltransferase HhaI with the aim of elucidating the molecular factors that explain why long-range DNA HT is inhibited when the glutamine residue inserted in the double helix is mutated into a tryptophan. We combine molecular dynamics, quantum chemistry, and kinetic Monte Carlo simulations and find that protein binding stabilizes the energies of the guanine radical cation states and significantly impacts the corresponding electronic couplings, thus determining the observed behavior, whereas the formation of a tryptophan radical leads to less efficient HT.

Epigenetic modifications are critical mechanisms that regulate many biological processes and establish normal cellular phenotypes. Aberrant epigenetic modifications are frequently linked to the development and maintenance of several diseases including cancer, inflammation and metabolic diseases and so on. The key proteins that mediate epigenetic modifications have been thus recognized as potential therapeutic targets for these diseases. Consequently, discovery of small molecule inhibitors for epigenetic targets has received considerable attention in recent years. Here, virtual screening methods and their applications in the discovery of epigenetic target inhibitors are the focus of this review. Newly emerging approaches or strategies including rescoring methods, docking pose filtering methods, machine learning methods and 3D molecular similarity methods were also underlined. They are expected to be employed for identifying novel inhibitors targeting epigenetic regulation more efficiently., (Copyright © 2014 Elsevier Inc. All rights reserved.)

DNA methylation is an epigenetic modification essential for normal development and maintenance of somatic biological functions. DNA methylation provides heritable, long-term chromatin regulation and the aberrant methylation pattern is associated with complex diseases including cancer. Discovering novel therapeutic targets demands development of high-throughput, sensitive and inexpensive screening platforms for libraries of chemical or biological matter involved in DNA methylation establishment and maintenance. Here, we present a universal, high-throughput, microfluidic-based fluorometric assay for studying DNA methylation in vitro. The enzymatic activity of bacterial HPAII DNA methyltransferase and its kinetic properties are measured using the assay (K(m)(DNA) = 5.8 nM, K(m)(SAM) = 9.8 nM and Kcat = 0.04 s(-1)). Using the same platform, we then demonstrate a two-step approach for high-throughput in vitro identification and characterization of small molecule inhibitors of methylation. The approach is examined using known non-nucleoside inhibitors, SGI-1027 and RG108, for which we measured IC50 of 4.5 μM and 87.5 nM, respectively. The dual role of the microfluidic-based methylation assay both for the quantitative characterization of enzymatic activity and high-throughput screening of non-nucleoside inhibitors coupled with quantitative characterization of the inhibition potential highlights the advantages of our system for epigenetic studies.

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) consists of a methyltransferase domain (residues 72-379) and an N-terminal region (residues 1-71) which regulates transcription in SsoII restriction-modification system. Small-angle X-ray scattering (SAXS) is employed here to study the low resolution structure of M.SsoII and its complex with DNA containing the methylation site. The shapes reconstructed ab initio from the SAXS data reveal two distinct protein domains of unequal size. The larger domain matches the crystallographic structure of a homologous DNA methyltransferase HhaI (M.HhaI), and the cleft in this domain is occupied by DNA in the model of the complex reconstructed from the SAXS data. This larger domain can thus be identified as the methyltransferase domain whereas the other domain represents the N-terminal region. Homology modeling of the M.SsoII structure is performed by using the model of M.HhaI for the methyltransferase domain and representing the N-terminal region either as a flexible chain of dummy residues or as a rigid structure of a homologous protein (phage 434 repressor) connected to the methyltransferase domain by a short flexible linker. Both models are compatible with the SAXS data and demonstrate high mobility of the N-terminal region. The linker flexibility might play an important role in the function of M.SsoII as a transcription factor.

DNA methyltransferases play crucial roles in establishing and maintenance of DNA methylation, which is an important epigenetic mark. Flipping the target cytosine out of the DNA helical stack and into the active site of protein provides DNA methyltransferases with an opportunity to access and modify the genetic information hidden in DNA. To investigate the conversion process of base flipping in the HhaI methyltransferase (M.HhaI), we performed different molecular simulation approaches on M.HhaI-DNA-S-adenosylhomocysteine ternary complex. The results demonstrate that the nonspecific binding of DNA to M.HhaI is initially induced by electrostatic interactions. Differences in chemical environment between the major and minor grooves determine the orientation of DNA. Gln237 at the target recognition loop recognizes the GCGC base pair from the major groove side by hydrogen bonds. In addition, catalytic loop motion is a key factor during this process. Our study indicates that base flipping is likely to be an "induced-fit" process. This study provides a solid foundation for future studies on the discovery and development of mechanism-based DNA methyltransferases regulators.

A method based on electrogenerated chemiluminescence (ECL) for detection of DNA methylation and assay of the methyltransferase activity is developed, and it is demonstrated that the label-free ECL method is capable of detecting methyltransferase with a detection limit of 3 × 10(-6) U mL(-1), using a supersandwich amplification technique.

DNA methylation is the most frequent form of epigenetic modification in the cell, which involves gene regulation in eukaryotes and protection against restriction enzymes in prokaryotes. Even though many methyltransferases exclusively modify their cognate sites, there have been reports of those that exhibit promiscuity. Previous experimental approaches used to characterize these methyltransferases do not provide the exact concentration at which off-target methylation occurs. Here, we present the first reported fidelity index (FI) for a number of DNA methyltransferases. We define the FI as the ratio of the highest amount of methyltransferase that exhibits no star activity (off-target effects) to the lowest amount that exhibits complete modification of the cognate site. Of the methyltransferases assayed, M.MspI and M.AluI exhibited the highest fidelity of ≥250 and ≥500, respectively, and do not show star activity even at very high concentrations. In contrast, M.HaeIII, M.EcoKDam and M.BamHI have the lowest fidelity of 4, 4 and 2, respectively, and exhibit star activity at concentrations close to complete methylation of the cognate site. The fidelity indexes provide vital information on the usage of methyltransferases and are especially important in applications where site specific methylation is required.

DNA cytosine methyltransferases regulate the expression of the genome through the precise epigenetic marking of certain cytosines with a methyl group, and aberrant methylation is a hallmark of human diseases including cancer. Targeting these enzymes for drug design is currently a high priority. We have utilized ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations to investigate extensively the reaction mechanism of the representative DNA methyltransferase HhaI (M.HhaI) from prokaryotes, whose overall mechanism is shared with the mammalian enzymes. We obtain for the first time full free energy profiles for the complete reaction, together with reaction dynamics in atomistic detail. Our results show an energetically preferred mechanism in which nucleophilic attack of cytosine C5 on the S-adenosyl-L-methionine (AdoMet) methyl group is concerted with formation of the Michael adduct between a conserved Cys in the active site with cytosine C6. Spontaneous and reversible proton transfer between a conserved Glu in the active site and cytosine N3 at the transition state was observed in our simulations, revealing the chemical participation of this Glu residue in the catalytic mechanism. Subsequently, the β-elimination of the C5 proton utilizes as base an OH(-) derived from a conserved crystal water that is part of a proton wire water channel, and this syn β-elimination reaction is the rate-limiting step. Design of novel cytosine methylation inhibitors would be advanced by our structural and thermodynamic characterization of the reaction mechanism.

A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180° around the deoxyribose-phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.

DNA methylation is an important epigenetic regulator of gene expression. Abnormalities in DNA methylation patterns have been associated with various developmental and proliferative diseases, particularly cancer. Targeting DNA methyltransferases (DNMTs) represents a promising strategy for the treatment of such diseases. Current DNMT inhibitors suffer important drawbacks with respect to their efficacy, specificity, and toxicity. In this study, we have set up a robust in vitro bacterial M.SssI DNMT activity assay to systematically screen a collection of 26 240 compounds that were predicted to compete with the S-adenosyl-L-methionine (SAM) substrate of DNMT. This resulted in the identification of a novel set of structurally distinct inhibitors of M.SssI DNMT activity. Although molecular docking studies using an M.SssI homology model suggest that these compounds might compete with SAM binding, mode of activity (MoA) assays are still needed to confirm this hypothesis. Our set of novel M.SssI DNMT inhibitors, once confirmed in an orthogonal DNMT assay, may thus serve as a starting point to identify and characterize suitable lead candidates for further drug optimization.

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA-M.HhaI-AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.

Background: How DNA 5-cytosine methyltransferases (DCMTases) select their substrate nucleobase for extrusion from DNA duplex is poorly understood., Results: The crystal structure of a pre-extrusion M.HaeIII DCMTase-substrate DNA complex is reported here., Conclusion: M.HaeIII selects its substrate cytosine for extrusion by selectively interfering with its stacking and hydrogen bonding interactions within the DNA duplex., Significance: This is the first structural elucidation of the target cytosine selection by a DCMTase. Epigenetic methylation of cytosine residues in DNA is an essential element of genome maintenance and function in organisms ranging from bacteria to humans. DNA 5-cytosine methyltransferase enzymes (DCMTases) catalyze cytosine methylation via reaction intermediates in which the DNA is drastically remodeled, with the target cytosine residue extruded from the DNA helix and plunged into the active site pocket of the enzyme. We have determined a crystal structure of M.HaeIII DCMTase in complex with its DNA substrate at a previously unobserved state, prior to extrusion of the target cytosine and frameshifting of the DNA recognition sequence. The structure reveals that M.HaeIII selects the target cytosine and destabilizes its base-pairing through a precise, focused, and coordinated assault on the duplex DNA, which isolates the target cytosine from its nearest neighbors and thereby facilitates its extrusion from DNA.

The human tRNA m ( 5) C methyltransferase Misu is a novel downstream target of the proto-oncogene Myc that participates in controlling cell division and proliferation. Misu catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosines in tRNAs. It was previously shown to catalyze in vitro the intron-dependent formation of m ( 5) C at the first position of the anticodon (position 34) within the human pre-tRNA (Leu) (CAA). In addition, it was recently reported that C48 and C49 are methylated in vivo by Misu. We report here the expression of hMisu in Escherichia coli and its purification to homogeneity. We show that this enzyme methylates position 48 in tRNA (Leu) (CAA) with or without intron and positions 48, 49 and 50 in tRNA (Gly2) (GCC) in vitro. Therefore, hMisu is the enzyme responsible for the methylation of at least four cytosines in human tRNAs. By comparison, the orthologous yeast enzyme Trm4 catalyzes the methylation of carbon 5 of cytosine at positions 34, 40, 48 or 49 depending on the tRNAs.

M.PvuII is a DNA methyltransferase from the bacterium Proteus vulgaris that catalyzes methylation of cytosine at the N4 position. This enzyme also displays promiscuous activity catalyzing methylation of adenine at the N6 position. In this work we use QM/MM methods to investigate the reaction mechanism of this promiscuous activity. We found that N6 methylation in M.PvuII takes place by means of a stepwise mechanism in which deprotonation of the exocyclic amino group is followed by the methyl transfer. Deprotonation involves two residues of the active site, Ser53 and Asp96, while methylation takes place directly from the AdoMet cofactor to the target nitrogen atom. The same reaction mechanism was described for cytosine methylation in the same enzyme, while the reversal timing, that is methylation followed by deprotonation, has been described in M.TaqI, an enzyme that catalyzes the N6-adenine DNA methylation from Thermus aquaticus. These mechanistic findings can be useful to understand the evolutionary paths followed by N-methyltransferases.

One step at a time: Substrates containing nucleotide analogues lacking sequence-specific contacts to the C5 methyltransferase M.HhaI were used to probe the role of individual interactions in effecting conformational transitions during base flipping. A segregation of duties, that is, specific recognition and chemomechanical force for base flipping and active site assembly, within the enzyme is confirmed., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Curcumin and resveratrol were evaluated for their potential to cause reversal of promoter hypermethylation and associated gene expression of FANCF in SiHa cell line. Methylation specific PCR along with bisulphite sequencing revealed the demethylation of 12 CpG sites out of 15 CpG sites spanning ?280 to ?432 region of FANCF promoter after treatment with curcumin and fivefold up regulation of FANCF gene expression as shown by qRT-PCR. In vitro methylation assay also showed that M.SssI an analogue of DNMT1 was effectively inhibited at 50 lM concentration of curcumin. Resveratrol was not found to be effective in causing reversal of promoter hypermethylation of FANCF gene when used at 20 lM for 4 days in SiHa cell line.

Background: Silencing mammalian genes by targeted DNA (cytosine-5) methylation of selected CG sites in the genome would be a powerful technique to analyze epigenomic information and to study the roles of DNA methylation in physiological and pathological states. A promising approach of targeted DNA methylation is based on the ability of split fragments of a monomeric DNA methyltransferase (C5-MTase) to associate and form active enzyme. A few C5-MTases of different specificities have been shown to possess the ability of fragment complementation, but a demonstration of this phenomenon for a C5-MTase, which has CG specificity and thus can be targeted to methylate any CG site, has been lacking. The purpose of this study was to test whether the CG-specific prokaryotic C5-MTase M.SssI shows the phenomenon of fragment complementation., Results: We show that truncated inactive N-terminal fragments of M.SssI can assemble with truncated inactive C-terminal fragments to form active enzyme in vivo when produced in the same E. coli cell. Overlapping and non-overlapping fragments as well as fragments containing short appended foreign sequences had complementation capacity. In optimal combinations C-terminal fragments started between conserved motif VIII and the predicted target recognizing domain of M.SssI. DNA methyltransferase activity in crude extracts of cells with the best complementing fragment pairs was ~ 4 per cent of the activity of cells producing the full length enzyme. Fusions of two N-terminal and two C-terminal fragments to 21.6 kDa zinc finger domains only slightly reduced complementation ability of the fragments., Conclusions: The CG-specific DNA methyltransferase M.SssI shows the phenomenon of fragment complementation in vivo in E. coli. Fusion of the split fragments to six unit zinc finger domains does not substantially interfere with the formation of active enzyme. These observations and the large number of complementing fragment combinations representing a wide range of MTase activity offer the possibility to develop M.SssI into a programmable DNA methyltransferase of high specificity.

The type II bifunctional DNA methyltransferase (MTase) Ecl18 that is able to control transcription of its own gene was studied kinetically. Based on initial velocity dependences from S-adenosyl-L-methionine (AdoMet) and target DNA and substrate preincubation assays, it is proposed that the enzyme apparently works by a rapid equilibrium ordered bi-bi mechanism with DNA binding first. By measuring the enzyme activity depending on DNA and AdoMet at different fixed concentrations of the operator sequence oligonucleotide, it was found that its binding has noncompetitive inhibitory effect on Ecl18 MTase activity.

The genome of Helicobacter pylori is rich in restriction-modification (RM) systems. Approximately 4% of the genome codes for components of RM systems. hpyAVIBM, which codes for a phase-variable C(5) cytosine methyltransferase (MTase) from H. pylori, lacks a cognate restriction enzyme. Over-expression of M.HpyAVIB in Escherichia coli enhances the rate of mutations. However, when the catalytically inactive F9N or C82W mutants of M.HpyAVIB were expressed in E. coli, mutations were not observed. The M.HpyAVIB gene itself was mutated to give rise to different variants of the MTase. M.HpyAVIB variants were purified and differences in kinetic properties and specificity were observed. Intriguingly, purified MTase variants showed relaxed substrate specificity. Homologues of hpyAVIBM homologues amplified and sequenced from different clinical isolates showed similar variations in sequence. Thus, hpyAVIBM presents an interesting example of allelic variations in H. pylori where changes in the nucleotide sequence result in proteins with new properties., (© 2012 The Authors Journal compilation © 2012 FEBS.)

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) functions as a methyltransferase and also as a transcription factor. Chemical and photochemical crosslinking was used for exploring the structure of M.SsoII-DNA complexes and M.SsoII in the absence of DNA. Photocrosslinking with 4-(N-maleimido)benzophenone demonstrated that in the M.SsoII complex with DNA containing the regulatory site, the M.SsoII region responsible for methylation was bound to DNA flanking the regulatory site, which contained no methylation sequence. This required high flexibility of the linker connecting the M.SsoII N-terminal domain and the M.SsoII region responsible for methylation. The flexibility was demonstrated by crosslinking with bis-maleimidoethane and 1,11-bis-maleimidotetraethyleneglycol.

Methylation of the five position of cytosine in DNA plays important roles in epigenetic regulation in diverse organisms including humans. The transfer of methyl groups from the cofactor S-adenosyl-L-methionine is carried out by methyltransferase enzymes. Using the paradigm bacterial methyltransferase M.HhaI we demonstrate, in a chemically unperturbed system, the first direct real-time analysis of the key mechanistic events-the flipping of the target cytosine base and its covalent activation; these changes were followed by monitoring the hyperchromicity in the DNA and the loss of the cytosine chromophore in the target nucleotide, respectively. Combined with studies of M.HhaI variants containing redesigned tryptophan fluorophores, we find that the target base flipping and the closure of the mobile catalytic loop occur simultaneously, and the rate of this concerted motion inversely correlates with the stability of the target base pair. Subsequently, the covalent activation of the target cytosine is closely followed by but is not coincident with the methyl group transfer from the bound cofactor. These findings provide new insights into the temporal mechanism of this physiologically important reaction and pave the way to in-depth studies of other base-flipping systems., (© The Author(s) 2011. Published by Oxford University Press.)

The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H(2)O(2)-mediated oxidation of 2,2'-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS(2-)) to a colored product ABTS(·-). This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis.

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5'-CCNGG-3' (N=A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5'-AGGACAAATTGTCCT-3' in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K(D), of the M.SsoII-DNA complexes decreased in the order 60oct>60reg>60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, k(a), was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell., (This journal is © The Royal Society of Chemistry 2011)

The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme-DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.

Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) has a long N-terminal region (1-71 residues) preceding the sequence with conservative motifs, which are characteristic for all DNA methyltransferases of such kind. The presence of this region provides M.SsoII capability to act as a transcription regulator in SsoII restriction-modification system. To perform its regulatory function, M.SsoII binds specifically to a 15-mer inverted repeat in the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein delta(72-379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII-like DNA-methyltransferase M.Ecl18kI and is homologous to M.SsoII N-terminal region. delta(72-379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of high protein excess relative to DNA, which could indicate an altered structure in the deletion mutant in comparison with the full-length M.SsoII. Circular dichroism spectroscopy demonstrated that delta(72-379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32% a-helices and 20% beta-sheets. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is made how alpha-helices and beta-sheets are arranged in M.SsoII N-terminal region.

Methylation of cytosine residues in the DNA is one of the most important epigenetic marks central to the control of differential expression of genes. We perform quantum mechanical calculations to investigate the catalytic mechanism of the bacterial HhaI DNA methyltransferase. We find that the enzyme nucleophile, Cys81, can attack C6 of cytosine only after it is deprotonated by the DNA phosphate group, a reaction facilitated by a bridging water molecule. This finding, which indicates that the DNA acts as both the substrate and the cofactor, can explain the total loss of activity observed in an analogous enzyme, thymidylate synthase, when the phosphate group of the substrate was removed. Furthermore, our results displaying the inability of the phosphate group to deprotonate the side chain of serine is in agreement with the total, or the large extent of, inactivity observed for the C81S mutant. In contrast to results from previous calculations, we find that the active site conserved residues, Glu119, Arg163, and Arg165, are crucial for catalysis. In addition, the enzyme-DNA adduct formation and the methyl transfer from the cofactor S-adenosyl-L-methionine are not concerted but proceed via stepwise mechanism. In many of the different steps of this methylation reaction, the transfer of a proton is found to be necessary. To render these processes possible, we find that several water molecules, found in the crystal structure, play an important role, acting as a bridge between the donating and accepting proton groups., (2010 Elsevier Ltd. All rights reserved.)

Oligonucleotides with 1,2-diol grouping were prepared from 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxo-ethyl]uridine 3'-phosphoramidite. The thermal stability of modified DNA duplexes and their ability to form complexes with the p50 subunit of the NF-kappaB transcription factor and (cytosine-5)-DNA methyltransferase SsoII were studied. The periodate oxidation of the l,2-diol grouping of the oligonucleotides resulted in reactive 2'-aldehyde derivatives. The opportunity of their use for the affinity modification of DNA-recognizing proteins was studied.

Using the type III restriction-modification enzyme EcoP15I, we isolated sequences flanking sites digested by the methylation-sensitive HpaII enzyme or its methylation-insensitive MspI isoschizomer for massively parallel sequencing. A novel data transformation allows us to normalise HpaII by MspI counts, resulting in more accurate quantification of methylation at >1.8 million loci in the human genome. This HELP-tagging assay is not sensitive to sequence polymorphism or base composition and allows exploration of both CG-rich and depleted genomic contexts.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.

DNA methylation affects important developmental processes in both plants and animals. The process of methylation of cytosines at C-5 is catalysed by DNA methyltransferases (MTases), which are highly conserved, both structurally and functionally, in eukaryotes. In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset of reproductive development in rice. The rice genome (Oryza sativa L. subsp. japonica) encodes a total of 10 genes that contain the highly conserved MTase catalytic domain. These genes have been categorized into subfamilies on the basis of phylogenetic relationships. A microarray-based gene expression profile of all 10 MTases during 22 stages/tissues that included 14 stages of reproductive development and five vegetative tissues together with three stresses, cold, salt and dehydration stress, revealed specific windows of MTase activity during panicle and seed development. The expression of six methylases was specifically/preferentially upregulated with the initiation of floral organs. Significantly, one of the MTases was also activated in young seedlings in response to cold and salt stress. The molecular studies presented here suggest a greater role for these proteins and the epigenetic process in affecting genome activity during reproductive development and stress than was previously anticipated.

Changes in the methylation pattern of genomic DNA, particularly hypermethylation of tumor suppressor genes, occur at early stages of tumor development. Errors in DNA methylation contribute to both initiation and progression of various cancers. This stimulates significant interest in searching for inhibitors of C5-DNA-methyltransferases (MTases). Here we review the known nucleoside mechanism-based reversible and irreversible inhibitors of the MTases, as well as non-nucleoside ones, and discuss their inhibitory mechanisms and application for MTase investigations and cancer therapy.

To characterize important steps of DNA methylation by M.SssI, a prokaryotic DNA-(cytosine C5)-methyltransferase (C5-MTase) sharing the specificity of eukaryotic C5-MTases (5'-CG-3'), ten amino acids, selected on the basis of sequence alignments and a computational model, were subjected to mutational analysis. Wild-type and mutant M.SssI variants were studied to determine methylation activity, DNA binding affinity, capacity to induce base flipping, and ability to form covalent complex with a DNA substrate containing the mechanism-based inhibitor 2-pyrimidinone. Wild-type M.SssI induced strong fluorescence when bound to substrate DNA containing 2-aminopurine in place of the target cytosine, indicating flipping of the target base. Reduced fluorescence, moderate, or drastic loss of methyltransferase activity and reduced DNA binding suggest the involvement of the conserved S145 (motif IV), R232 (motif VIII, QxRxR), and T313 (variable region, conserved TL), as well as of the non-conserved Q147 in base flipping. Replacement of E186 (motif VI, ENV) and R230 (motif VIII, QxRxR) with alanine resulted in loss of methyltransferase activity without impairing DNA binding affinity. These data are consistent with the catalytic role of E186 and R230, and provide, for the first time, experimental support for the essential function of the hitherto not investigated invariant arginine of motif VIII in C5-MTases.

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases.

Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M.HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme-cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed. Furthermore, switching from unmethylated to hemimethylated cognate DNA involves detectable protein conformational changes 20-30 A away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.