Investigation Of The Methylation Mechanisms By DNMT2

DNA methylation at the C5-position of cytosine,catalyzed by DNA methyltransferases(DNMTs),is the predominant epigenetic modification in mammals.Mammalian DNMTs include DNMT1,DNMT2 and DNMT3.DNMT1 and DNMT3 are responsible for the maintenance of DNA methylation patterns and de novo DNA methylation,respectively.DNMT2 only exhibits very weak DNA methylation activity in vitro.Whether DNMT2 methylates DNA is still controversial.In 2006,Homo sapiens DNMT2(hDNMT2)was discovered to mainly methylate cytosine 38 of eukaryotic t RNAAsp(GUC).Currently,the known t RNA substrates of eukaryotic DNMT2 also include t RNAGly(GCC)and t RNAVal(CAC).A recent study reported that t RNAGlu(UUC)from Geobacter sulfurreducens is also a substrate of Gs DNMT2.Nevertheless,how DNMT2 recognizes t RNA substrates is unclear.In this study,in an effort to optimize the in vitro methylation buffer for human DNMT2,the effect of disulfide bond formation on the structure of hDNMT2 and its in vitro t RNA methylation activity was investigated.In addition,the effect of increased GSH or GSSG on the localization and expression of DNMT2 in cells was also examined.The preference of hDNMT2 to different eukaryotic t RNA substrates and the important determinants that affect the methylation activity of hDNMT2 in t RNA structure were identified.The in vitro DNA methylation activity of hDNMT2 was also discussed.Finally,the key functional domains of hDNMT2 for the selection of substrate t RNA and DNA were explored.The main results are as follows:1.Optimization of methylation condition of hDNMT2 in vitroWhile optimizing the concentrations of Mg Cl2,EDTA,DTT and identifying the optimal salt type and concentration,the t RNA methylation activity of hDNMT2 was optimal in a methylation buffer containing 50 m M Tris(p H 7.5),50 m M NH4 OAc,1 m M Mg Cl2,0.1 m M EDTA and 6 m M DTT at 37 ° C.The methylation activity of hDNMT2 was highly sensitive to DTT concentration,so It is speculated that disulfide bonds may exist in hDNMT2 protein purified.2.Formation of intermolecular disulfide bonds in hDNMT2The structural analysis of modeled DNMT2 homologous indicated that the cysteines in hDNMT2 had little chance of forming an intramolecular disulfide bond.By using Native-PAGE electrophoresis,it was found that the reducing concentration of DTT resulted in the formation of hDNMT2 polymers.So,intermolecular disulfide bonds could be formed in hDNMT2 proteins purified.Using LC-MS/MS analysis,hDNMT2 dimer was found in purified hDNMT2 using 2.5 mm DTT.The dimer was stabilized by a intermolecular disulfide bond C292-C292.The reduction of DTT concentration to 1 m M further resulted in the formation of additional disulfide bonds.The formation of these additional disulfide bonds may be the consequence of misfolding and aggregation of hDNMT2 at 2.5 m M or lower concentrations of DTT.In cells,GSH plays an important role in breaking disulfide bonds and maintaining free sulfhydryl groups of cysteine residues.CD spectra revealed that similar significant conformational changes of hDNMT2 caused by the concentration change of DTT or GSH.HEK293 T growth was inhibited by stress condition induced by GSH or GSSG,which increased the cytoplasmic localization of hDNMT2,though the nucleus localization was still dominant.GSH could break the intermolecular disulfide bond of hDNMT2 and increased the methylation activites of t RNA substrates in cells.3.Recognition mechanism of t RNA methylation and a new t RNA substrateThe methylation activities of hDNMT2 decreased with these four t RNA substrates in the order of t RNAGly,t RNAVal,t RNAAspand t RNAGlu,suggesting that the preferred substrate of hDNMT2 in vitro was t RNAGly.Hence,CD spectrum of the deletion mutants and the point mutants of human t RNAGlywere detected,and methylation activitives of hDNMT2 on t RNAGlymutants were analyzed.These data implied that the bases in anticodon loop were in the active site and these bases in D-loop and T-loop play a critical role in maintaining t RNA tertiary structure.What is more,the important determinants in t RNAGlywere identified for hDNMT2 activity.To further verify the importance of the components,t RNAs were picked as they contain some similar determinants.Surprisingly,weak methylation activity of hDNMT2 on t RNAGln(CTG)was found in vitro.Some important determinants affecting the methylation activity of hDNMT2 in the t RNA structure has great potential value in modifying the bases of RNA aptamer and extending the half-life of drugs.4.DNA methylation mechanism of DNMT2The binding of hDNMT2 to a specific DNA sequence was predicted by the analysis of the surface potential of hDNMT2 structure.The dynamic simulation of the complex structure of hDNMT2 and DNA further revealed the interaction mechanism of hDNMT2 and DNA.The binding ability of hDNMT2 to the DNA fragment was confirmed by agarose gel electrophoresis/digestion assay,which could effectively prevent the endonuclease from cutting the DNA fragment.In vitro methylation tests showed that hDNMT2 could methylate this DNA fragment.The DNA methylation by hDNMT2 needs further investigation in vivo,and the detailed methylation mechanism still remains to be addressed.5.The domains in DNMT2 affecting the substrate selectionTRD1 and TRD2 domains emerged in the DNMT2 homologous sequences during evolution,and played a key role in the selection of nucleic acid(t RNA or DNA)substrate of DNMT2 by bioinformatical methods and in vitro enzymatic activity tests.S219,S223,G224,I228,L229 and G305 in TRD1 and TRD2 regions play an important role in promoting the selection of t RNA substrates of hDNMT2.The preference of DNMT2 to t RNA may be related to the TRD1 domain by sequence alignment,hydrophilic degree and surface potential distribution.The TRD2 domain is a region with high incidences of cancerous variations.The cancerous variants occurred in the TRD2 domain significantly affected the methylation activity of hDNMT2.The important residues and domains that affect the methylation activity of hDNMT2 have been found to be of great significance for the development of effective antitumor drugs,antiviral drugs and antiparasitic drugs.