Biochemical and structural characterization of the first-discovered metazoan DNA cytosine-N4 methyltransferase from the bdelloid rotifer Adineta vaga.

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.

Label-free and near-zero-background-noise photoelectrochemical assay of methyltransferase activity based on a Bi2S3/Ti3C2 Schottky junction.

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 Bi2S3/Ti3C2 nanosheets. The proposed PEC sensor exhibited a low detection limit and a high signal-to-noise ratio for M.SssI MTase assay.

A novel signal-off photoelectrochemical biosensor for M.SssI MTase activity assay based on GQDs@ZIF-8 polyhedra as signal quencher.

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 TiO2, poly(diallyldimethylammonium chloride) and CdTe quantum dots (QDs). The obtained electrode was used as the photoelectrode and labeled as ITO/TiO2/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/TiO2/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.

Single-Molecule Fluorescence Imaging for Ultrasensitive DNA Methyltransferase Activity Measurement and Inhibitor Screening.

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. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, 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. SssI 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. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Role of Conformational Fluctuations of Protein toward Methylation in DNA by Cytosine-5-methyltransferase.

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.

Mirror Bisulfite Sequencing: A Method for Single-Base Resolution of Hydroxymethylcytosine.

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 ß-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.

Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit.

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.

Sensitive fluorescent detection of methyltransferase based on thermosensitive poly(N-isopropylacrylamide).

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'-CmCGG-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.

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase.

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.

Characterization of Vsr endonucleases from Neisseria meningitidis.

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.

Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

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.

Identification of the methyltransferase targeting C2499 in Deinococcus radiodurans 23S ribosomal RNA.

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.

A 7-Deazaadenosylaziridine Cofactor for Sequence-Specific Labeling of DNA by the DNA Cytosine-C5 Methyltransferase M.HhaI.

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.

Single Amino Acid Mutation Controls Hole Transfer Dynamics in DNA-Methyltransferase HhaI Complexes.

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.

Virtual screening in small molecule discovery for epigenetic targets.

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.

Metadynamics simulation study on the conformational transformation of HhaI methyltransferase: an induced-fit base-flipping hypothesis.

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 sensitive microfluidic platform for a high throughput DNA methylation assay.

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.

Flexibility of the linker between the domains of DNA methyltransferase SsoII revealed by small-angle X-ray scattering: implications for transcription regulation in SsoII restriction-modification system.

(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.

Fidelity index determination of DNA methyltransferases.

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.

A label-free supersandwich electrogenerated chemiluminescence method for the detection of DNA methylation and assay of the methyltransferase activity.

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.