Mechanisms and Biological Roles of DNA Methyltransferases and DNA Methylation: From Past Achievements to Future Challenges.

DNA methylation and DNA methyltransferases (MTases)-the enzymes that introduce the methylation mark into the DNA-have been studied for almost 70 years. In this chapter, we review the key developments in the DNA methylation field that have led to our current understanding of the structures and mechanisms of DNA MTases. We discuss the essential biological roles of DNA methylation, including the discovery of DNA methylation, cloning and sequence analysis of the bacterial and eukaryotic MTases, and the elucidation of their structure, mechanism, regulation, and molecular evolution. We describe genetic studies that contributed greatly to the evolving views on the role of DNA methylation in development and diseases, the invention of methods for the genome-wide analysis of DNA methylation, and the biochemical identification of DNA MTases and the TET enzyme family, which is involved in DNA demethylation. We summarize the roles of MTases in bacterial epigenetics and the application of MTases in synthetic biology to generate artificial signaling systems. We finish by highlighting some open questions for the next years of research in the field.

DNA Labeling Using DNA Methyltransferases.

DNA methyltransferases (MTases) uniquely combine the ability to recognize and covalently modify specific target sequences in DNA using the ubiquitous cofactor S-Adenosyl-L-methionine (AdoMet). Although DNA methylation plays important roles in biological signaling, the transferred methyl group is a poor reporter and is highly inert to further biocompatible derivatization. To unlock the biotechnological power of these enzymes, extended cofactor AdoMet analogs have been developed that enable targeted MTase-directed attachment of larger moieties containing functional or reporter groups onto DNA. As the enlarged cofactors are not always compatible with the active sites of native MTases, steric engineering of the active site has been employed to optimize their alkyltransferase activity. In addition to the described cofactor analogs, recently discovered atypical reactions of DNA cytosine-5 MTases involving non-cofactor-like compounds can also be exploited for targeted derivatization and labeling of DNA. Altogether, these approaches offer new powerful tools for sequence-specific covalent DNA labeling, leading to a variety of useful techniques in DNA research, diagnostics and nanotechnologies, and have already proven practical utility for optical DNA mapping and high-throughput epigenome studies.

7-Aminoalkoxy-Quinazolines from Epigenetic Focused Libraries Are Potent and Selective Inhibitors of DNA Methyltransferase 1.

Inhibitors of epigenetic writers such as DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug and probe discovery. To advance epigenetic probes and drug discovery, chemical companies are developing focused libraries for epigenetic targets. Based on a knowledge-based approach, herein we report the identification of two quinazoline-based derivatives identified in focused libraries with sub-micromolar inhibition of DNMT1 (30 and 81 nM), more potent than S-adenosylhomocysteine. Also, both compounds had a low micromolar affinity of DNMT3A and did not inhibit DNMT3B. The enzymatic inhibitory activity of DNMT1 and DNMT3A was rationalized with molecular modeling. The quinazolines reported in this work are known to have low cell toxicity and be potent inhibitors of the epigenetic target G9a. Therefore, the quinazoline-based compounds presented are attractive not only as novel potent inhibitors of DNMTs but also as dual and selective epigenetic agents targeting two families of epigenetic writers.

Structural and functional diversity among Type III restriction-modification systems that confer host DNA protection via methylation of the N4 atom of cytosine.

We report a new subgroup of Type III Restriction-Modification systems that use m4C methylation for host protection. Recognition specificities for six such systems, each recognizing a novel motif, have been determined using single molecule real-time DNA sequencing. In contrast to all previously characterized Type III systems which modify adenine to m6A, protective methylation of the host genome in these new systems is achieved by the N4-methylation of a cytosine base in one strand of an asymmetric 4 to 6 base pair recognition motif. Type III systems are heterotrimeric enzyme complexes containing a single copy of an ATP-dependent restriction endonuclease-helicase (Res) and a dimeric DNA methyltransferase (Mod). The Type III Mods are beta-class amino-methyltransferases, examples of which form either N6-methyl adenine or N4-methyl cytosine in Type II RM systems. The Type III m4C Mod and Res proteins are diverged, suggesting ancient origin or that m4C modification has arisen from m6A MTases multiple times in diverged lineages. Two of the systems, from thermophilic organisms, required expression of both Mod and Res to efficiently methylate an E. coli host, unlike previous findings that Mod alone is proficient at modification, suggesting that the division of labor between protective methylation and restriction activities is atypical in these systems. Two of the characterized systems, and many homologous putative systems, appear to include a third protein; a conserved putative helicase/ATPase subunit of unknown function and located 5' of the mod gene. The function of this additional ATPase is not yet known, but close homologs co-localize with the typical Mod and Res genes in hundreds of putative Type III systems. Our findings demonstrate a rich diversity within Type III RM systems.

A Mutation-Based Method for Pinpointing a DNA N6 -Methyladenine Methyltransferase Modification Site at Single Base Resolution.

DNA N6 -methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme-assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl-substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N6 -position of adenine within a specific DNA sequence to form N6 -allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N6 -cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA-containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single-base resolution.

Role of traditional CHO PET parameters in distinguishing IDH, TERT and MGMT alterations in primary diffuse gliomas.

OBJECTIVE: Isocitrate dehydrogenase (IDH) mutation, telomerase reverse transcriptase (TERT) promoter mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status are diagnostic, prognostic, predictive and therapeutic biomarkers for primary diffuse gliomas, and this study aimed to explore the relationship between choline (CHO) positron emission tomography (PET) parameters and these molecular alterations. METHODS: Twenty-eight patients who were histopathologically diagnosed with primary diffuse glioma and underwent presurgical CHO PET/CT were retrospectively analyzed, and IDH, TERT and MGMT alterations were examined. The volume of interest (VOI) was semiautomatically defined based on standardized uptake value (SUV) thresholds, and 5 traditional CHO parameters, namely, SUVmax, SUVmean, metabolic tumor volume (MTV), total lesion CHO uptake (TLC) and tumor-to-normal contralateral cortex activity ratio (T/N ratio), were calculated. Wilcoxon rank-sum tests and receiver operating characteristic (ROC) curves were applied to evaluate the differences and performances of the CHO parameters, and their capability to stratify patient prognosis was also evaluated. RESULTS: All 5 parameters were significantly higher in IDH-wildtype gliomas than in IDH-mutant gliomas (p = 0.0001-0.037), and SUVmax, SUVmean, TLC and the T/N ratio exhibited good performances in distinguishing the IDH status (areas under the ROC curve (AUCs) 0.856-0.918, accuracies 0.857-0.893) as well as stratifying patient prognosis. Although the differences and performances of the traditional parameters in distinguishing diverse TERT and MGMT statuses were moderate in the whole population, the T/N ratio and TLC displayed certain predictive value in discriminating the TERT status in the IDH-mutant and IDH-wildtype subgroups (p = 0.028-0.048, AUCs 0.857-0.860, accuracies 0.800-0.917, respectively). CONCLUSIONS: Traditional CHO PET parameters are capable of distinguishing IDH but not TERT or MGMT alterations in the whole population. In accordance with the clinical understanding of TERT promoter mutations, the T/N ratio and TLC can also discriminate the TERT status in IDH subgroups.

Comprehensive analysis of regulation of DNA methyltransferase isoforms in human breast tumors.

Significant reprogramming of epigenome is widely described during pathogenesis of breast cancer. Transformation of normal cell to hyperplastic cell and to neoplastic phenotype is associated with aberrant DNA (de)methylation, which, through promoter and enhancer methylation changes, activates oncogenes and silence tumor suppressor genes in variety of tumors including breast. DNA methylation, one of the major epigenetic mechanisms is catalyzed by evolutionarily conserved isoforms namely, DNMT1, DNMT3A and DNMT3B in humans. Over the years, studies have demonstrated intricate and complex regulation of DNMT isoforms at transcriptional, translational and post-translational levels. The recent findings of allosteric regulation of DNMT isoforms and regulation by other interacting chromatin modifying proteins emphasizes functional integrity and their contribution for the development of breast cancer and progression. DNMT isoforms are regulated by several intrinsic and extrinsic parameters. In the present review, we have extensively performed bioinformatics analysis of expression of DNMT isoforms along with their transcriptional and post-transcriptional regulators such as transcription factors, interacting proteins, hormones, cytokines and dietary elements along with their significance during pathogenesis of breast tumors. Our review manuscript provides a comprehensive understanding of key factors regulating DNMT isoforms in breast tumor pathology and documents unsolved issues.

Engineered SAM Synthetases for Enzymatic Generation of AdoMet Analogs with Photocaging Groups and Reversible DNA Modification in Cascade Reactions.

Methylation and demethylation of DNA, RNA and proteins has emerged as a major regulatory mechanism. Studying the function of these modifications would benefit from tools for their site-specific inhibition and timed removal. S-Adenosyl-L-methionine (AdoMet) analogs in combination with methyltransferases (MTases) have proven useful to map or block and release MTase target sites, however their enzymatic generation has been limited to aliphatic groups at the sulfur atom. We engineered a SAM synthetase from Cryptosporidium hominis (PC-ChMAT) for efficient generation of AdoMet analogs with photocaging groups that are not accepted by any WT MAT reported to date. The crystal structure of PC-ChMAT at 1.87 Šrevealed how the photocaged AdoMet analog is accommodated and guided engineering of a thermostable MAT from Methanocaldococcus jannaschii. PC-MATs were compatible with DNA- and RNA-MTases, enabling sequence-specific modification ("writing") of plasmid DNA and light-triggered removal ("erasing").

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate.

SNAP-tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ® and its relative thermostable version (SsOGT-H5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

A novel Cas9-targeted long-read assay for simultaneous detection of IDH1/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma.

Molecular biomarkers provide both diagnostic and prognostic results for patients with diffuse glioma, the most common primary brain tumor in adults. Here, we used a long-read nanopore-based sequencing technique to simultaneously assess IDH mutation status and MGMT methylation level in 4 human cell lines and 8 fresh human brain tumor biopsies. Currently, these biomarkers are assayed separately, and results can take days to weeks. We demonstrated the use of nanopore Cas9-targeted sequencing (nCATS) to identify IDH1 and IDH2 mutations within 36 h and compared this approach against currently used clinical methods. nCATS was also able to simultaneously provide high-resolution evaluation of MGMT methylation levels not only at the promoter region, as with currently used methods, but also at CpGs across the proximal promoter region, the entirety of exon 1, and a portion of intron 1. We compared the methylation levels of all CpGs to MGMT expression in all cell lines and tumors and observed a positive correlation between intron 1 methylation and MGMT expression. Finally, we identified single nucleotide variants in 3 target loci. This pilot study demonstrates the feasibility of using nCATS as a clinical tool for cancer precision medicine.

Systematic Analysis of the DNA Methylase and Demethylase Gene Families in Rapeseed (Brassica napus L.) and Their Expression Variations After Salt and Heat stresses.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

A protein architecture guided screen for modification dependent restriction endonucleases.

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase.

DNA methyltransferase (MTase) and polynucleotide kinase (PNK) are both DNA-dependent enzymes that play important roles in DNA methylation and DNA repair processes, respectively. Dysregulation of their activities is associated with various human diseases. Herein, we present a specific and sensitive biosensing strategy, named terminal deoxynucleotidyl transferase (TdT)-activated nicking enzyme amplification reaction (TdT-NEAR), for their activity detection. As for MTase detection, an enclosed dumbbell-shaped oligonucleotide substrate, whose symmetric stem containing a recognition site of Dam MTase and an incomplete recognition sequence of nicking endonuclease Nt.BbvCI, was used. Typically, the substrate is methylated by Dam MTase and subsequently cleaved by Dpn I. In the presence of TdT and dGTP, poly(guanine, G) sequences are extended from the released 3'-OH ends, achieving the conversion of the incomplete Nt.BbvCI recognition sequence to an intact one. The extension products can then be used to trigger Nt.BbvCI-catalyzed cyclic cleavage of fluorophore/quencher-labelled oligonucleotide probe, giving a significantly enhanced fluorescence output. Such a sensing system can achieve sensitive and specific detection of Dam MTase with a detection limit of 0.002 U/mL. The unique working mechanism endows the sensing system with improved anti-interference capability and thus increased application potential in complex biological samples. Moreover, it was also demonstrated to work well for Dam MTase inhibitor screening and inhibitory activity evaluation, thus holding great potential in disease diagnosis and drug discovery. Using a simpler 3'-phosphorylated linear substrate and the same fluorescent probe, the TdT-NEAR strategy can be easily extended to the activity analysis of PNK, thus revealing wide application potential in bioanalysis.

Quercetin modifies 5'CpG promoter methylation and reactivates various tumor suppressor genes by modulating epigenetic marks in human cervical cancer cells.

The central role of epigenomic alterations in carcinogenesis has been widely acknowledged, particularly the impact of DNA methylation on gene expression across all stages of carcinogenesis is considered vital for both diagnostic and therapeutic strategies. Dietary phytochemicals hold great promise as safe anticancer agents and effective epigenetic modulators. This study was designed to investigate the potential of a phytochemical, quercetin as a modulator of the epigenetic pathways for anticancer strategies. Biochemical activity of DNA methyltransferases (DNMTs), histone deacetylases (HDACs), histone methyltransferases (HMTs), and global genomic DNA methylation was quantitated by an enzyme-linked immunosorbent assay based assay in quercetin-treated HeLa cells. Molecular docking studies were performed to predict the interaction of quercetin with DNMTs and HDACs. Quantitative methylation array was used to assess quercetin-mediated alterations in the promoter methylation of selected tumor suppressor genes (TSGs). Quercetin induced modulation of chromatin modifiers including DNMTs, HDACs, histone acetyltransferases (HAT) and HMTs, and TSGs were assessed by quantitative reverse transcription PCR (qRT-PCR). It was found that quercetin modulates the expression of various chromatin modifiers and decreases the activity of DNMTs, HDACs, and HMTs in a dose-dependent manner. Molecular docking results suggest that quercetin could function as a competitive inhibitor by interacting with residues in the catalytic cavity of several DNMTs and HDACs. Quercetin downregulated global DNA methylation levels in a dose- and time-dependent manner. The tested TSGs showed steep dose-dependent decline in promoter methylation with the restoration of their expression. Our study provides an understanding of the quercetin's mechanism of action and will aid in its development as a candidate for epigenetic-based anticancer therapy.

MGMT autoantibodies as a potential prediction of recurrence and treatment response biomarker for glioma patients.

BACKGROUND: Cancer-specific autoantibodies found in serum of cancer patients have been characterized as potential predictors of the high risk of recurrence and treatment response. The objective of this study is to investigate the clinical utility of serum O-6-methylguanine-DNA methyltransferase (MGMT) autoantibodies as novel biomarkers for prediction of recurrence and treatment response for glioma through MGMT peptides microarray. METHODS: A total of 201 serum samples of glioma patients with various WHO grade and 311 serum samples of healthy donors were examined for the detection of MGMT autoantibodies by peptides microarray. The clinical value of MGMT autoantibodies was studied through univariable and multivariable analyses. RESULTS: Autoantibodies to MGMT peptides were detected in sera from glioma patients and five highly responsive autoantibodies to peptides were identified in the glioma group. The positive rate of MGMT autoantibody to 20 peptides in glioma groups is compared with healthy individuals, the positive rate of MGMT-02 (45%), MGMT-04 (27%), MGMT-07 (21%), MGMT-10 (13%), and MGMT-18 (24%) were significantly elevated in patients with glioma. MGMT autoantibody and its protein expression exhibited a significant correlation. The levels of MGMT autoantibodies decreased on the 30th day after operation, reaching preoperative levels, similar to those when tumor recurrence developed. Univariable and multivariable analyses revealed that the only preoperative autoantibodies to MGMT-02 peptide were independently correlated with recurrence-free survival. Preoperative seropositive patients were more likely than seronegative patients to have shorter recurrence times and to be resistant to chemoradiotherapy or chemotherapy with temozolomide. CONCLUSION: Monitoring the levels of preoperative serum autoantibodies to MGMT-02 peptide was useful for predicting patients at high risk of recurrence and treatment response.

Kinetic and catalytic properties of M.HpyAXVII, a phase-variable DNA methyltransferase from Helicobacter pylori.

The bacterium Helicobacter pylori is one of the most common infectious agents found in the human stomach. H. pylori has an unusually large number of DNA methyltransferases (MTases), prompting speculation that they may be involved in the cancerization of epithelial cells. The mod-4a/4b locus, consisting of the hp1369 and hp1370 ORFs, encodes for a truncated and inactive MTase in H. pylori strain 26695. However, slipped-strand synthesis within the phase-variable polyguanine tract in hp1369 results in expression of an active HP1369-1370 fusion N6-adenine methyltransferase, designated M.HpyAXVII. Sequence analysis of the mod-4a/4b locus across 74 H. pylori strain genomes has provided insights into the regulation of M.HpyAXVII expression. To better understand the role of M.HpyAXVII in the H. pylori biology, here we cloned and overexpressed the hp1369-70 fusion construct in Escherichia coli BL21(DE3) cells. Results from size-exclusion chromatography and multi-angle light scattering (MALS) analyses suggested that M.HpyAXVII exists as a dimer in solution. Kinetic studies, including product and substrate inhibition analyses, initial velocity dependence between substrates, and isotope partitioning, suggested that M.HpyAXVII catalyzes DNA methylation in an ordered Bi Bi mechanism in which the AdoMet binding precedes DNA binding and AdoMet's methyl group is then transferred to an adenine within the DNA recognition sequence. Altering the highly conserved catalytic motif (DPP(Y/F)) as well as the AdoMet-binding motif (FXGXG) by site-directed mutagenesis abolished the catalytic activity of M.HpyAXVII. These results provide insights into the enzyme kinetic mechanism of M.HpyAXVII. We propose that AdoMet binding conformationally "primes" the enzyme for DNA binding.

Local delivery of temozolomide via a biologically inert carrier (Temodex) prolongs survival in glioma patients, irrespectively of the methylation status of MGMT.

Glioma is the most common brain malignancy. Standard first-line therapy for glioma includes surgery, radiotherapy and systemic administration of temozolomide. However, temozolomide does not reach the brain in sufficient doses when administered orally and has poor efficiency in more than half of the patients. Strategies to improve the treatment of glial malignancies are therefore needed. We have recently developed a system (Temodex) for local administration of temozolomide by encapsulating the drug in a biologically inert matrix. Here, we assessed the effect of Temodex in combination with standard therapy in a small-scale clinical study. Since the efficacy of temozolomide therapy is known to depend on the methylation status of the O6-methylguanine-DNA methyltransferase gene (MGMT) promoter, we also analyzed whether the effect of Temodex was influenced by the methylation status of MGMT. Our data show that the combination of standard therapy and Temodex was more efficient than standard therapy alone, promoting the overall patient survival by up to 33 weeks. Moreover, the efficacy of Temodex was not dependent on the methylation status of MGMT. Local Temodex administration in combination with standard therapy thereby emerges as a novel therapeutic option, with applicability that is independent on the methylation status of the MGMT promoter.

Bio-analytical applications of nicking endonucleases assisted signal-amplification strategies for detection of cancer biomarkers -DNA methyl transferase and microRNA.

The low concentrations of cancer biomarkers in the blood have limited the utility of quantitative bioassays developed for the purpose. The advent of nicking endonucleases (NEases) as signal amplification tools have greatly enhanced the detection efficiency and provided a multi-optional platform to design target specific detection methods. The present review focuses on the prominent features of NEases, modified DNA probes (such as hairpin (HP) probes, molecular beacons, and G- quadruplex) that mediate cyclic cascade and role of helper enzymes. Application of NEase assisted signal amplification (NESA) has been discussed for diagnosis of two prominent cancer biomarkers viz. DNA methyl transferase (Dam MTase) and microRNA (miRNA). NESA mediated techniques such as rolling circle amplification (RCA), strand displacement amplification (SDA) and isothermal exponential amplification (EXPAR), have been compared in light of their future applications in clinical diagnosis. Significance of nanomaterials to achieve further amplification and NESA assays for simultaneous detection of miRNAs has also been conversed. It is anticipated that the information gained from the analyses of the prospects and limitations of NESA-based assays will be useful towards understanding the applications, and improvement of efficient isothermal exponential amplification strategies for highly sensitive and selective detection of cancer biomarkers.

MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP.

Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.

Genome-wide identification, evolution of DNA methyltransferases and their expression during gonadal development in Nile tilapia.

DNA methyltransferases (dnmts) are responsible for DNA methylation and play important roles in organism development. In this study, seven dnmts genes (dnmt1, dnmt2, dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1, dnmt3bb.2) were identified in Nile tilapia. Comprehensive analyses of dnmts were performed using available genome databases from representative animal species. Phylogenetic analysis revealed that the dnmts family were highly conserved in teleosts. Based on transcriptome data from eight adult tilapia tissues, the dnmts were found to be dominantly expressed in the head kidney, testis and ovary. Analyses of the gonadal transcriptome data in different developmental stages revealed that all dnmts were expressed in both ovary and testis, and four de novo dnmts (dnmt3aa, dnmt3ab, dnmt3bb.1, dnmt3bb.2) showed higher expression in the testis than in the ovary. Furthermore, during sex reversal induced by Fadrozole, the expression of these four de novo dnmts increased significantly in treated group compared to female control group. By in situ hybridization, the seven dnmts were found to be expressed mainly in phase I and II oocytes of the ovary and spermatocytes of the testis. When gonads were incubated with a methyltransferase inhibitor (5-AzaCdR) in vitro, the expression of dnmts genes were down-regulated significantly, while the expression of cyp19a1a (a key gene in female pathway) and dmrt1 (a key gene in male pathway) increased significantly. Our results revealed the conservation of dnmts during evolution and indicated a potential role of dnmts in epigenetic regulation of gonadal development.