Theoretical Studies On Glycosylases-Catalyzed DNA Demethylation

DNA demethylation is a crucial chemical process for the growth and development of living organisms.This process can dynamically adjust the genome methylation pattern,programmatically regulate the expression of related proteins.DNA demethylation is triggered by the DNA glycosylases,which are identifying and removing damaged base from the DNA backbone,by catalyzing the cleavage of the N-glycosidic bond between the damaged base and deoxyribose.Abnormal glycosylase function is associated with cell senescence,embryo death and cancer.The current knowledge of DNA demethylation is limited to the characterization of epigenetic experiments.Therefore,understanding the chemical mechanism of DNA demethylation and its regulation in gene expression catalyzed by glycosylases at molecular level.Which has great scientific significance for revealing the rules of DNA epigenetic inheritance,recognizing the development regulation of early life and promoting the prevention and treatment of diseases.In this paper,molecular dynamics(MD)simulation and quantum mechanics/molecular mechanics(QM/MM)were employed to explore the excision mechanism of 5-carboxycytosine(5-ca C)and 3-methyladenine(3m A)by two types of glycosylases:(1)Thymidine DNA glycosylase(TDG)catalyze 5-carboxycytosine excision:In this part,four types of 5-carboxycytosine were constructed and the TDG excision mechanism was studied in detail.TDG has a wide range of catalytic activity,that can specifically remove 5-methylcytosine(5-m C)oxidation derivative 5-ca C,and complete the demethylation process.The reaction follows a concerted mechanism(S_N2),with a single oxacarbenium ion-like transition state(TS).The substrate is activated through rotation around the phosphate backbone before the glycosidic bond cleavage,the substrate rearrangement diminishs the electrostatic repulsion between the anionic base and the phosphate backbone,resulting in a decrease in energy barrier.The energy barriers of TDG excise monoanion and different protonated forms 5-ca C is anion>amino>imino>zwitterion.The reaction involves acid catalysis,the monoanion form 5-ca C is difficult to be hydrolyzed,but the protonated forms are relatively favorable.Besides,the Thr197 and Asn140 residues have crucial role in positioning nucleophile molecule,electrostatic stabilizing the positive charge,promoting proton transfer,etc.(2)Alk D glycosylase catalyze 3-methyladenine excision:Alk D excises cationic damage by a non-base-flipping mechanism,and 3m A remains stacked in the duplex over the course of the reaction.Catalytic reaction follows a concerted mechanism,the N-glycosidic bond hydrolysis simultaneously with the nucleophile addition,and then a nucleophile proton transferred to the Asp113.Weak interaction analysis show that Alk D does not contact directly with the base,the Trp187 and Trp109 promote glycosidic bond cleavage through C-H/?interactions with deoxyribose.Residue mutation analysis show that the barriers are significantly increased,confirming the important role of Trp residues in the reaction process.In addition,the specificity of Alk D for cationic modification was demonstrated by excision studies of 3d3m A.The above computational studies will provide theoretical reference for clarifying the process of DNA demethylation and exploring the possible pathway of demethylation reaction.