Study On The Structure And Function Of DNA 6mA Methyltransferase MTA1

DNA methylation is an important epigenetic modification,mainly include 5-methylcytosine(5mC)and N6-methyladenine(6mA).Extensive research in mammalian is 5mC,and that of prokaryotes is 6mA.In recent years,6mA modification of DNA has been increasingly discovered in eukaryotes,and it is involved in many important biological processes such as gene expression regulation,nucleosome localization,transgenerational inheritance,and embryonic development.Researchers identified the DNA 6mA methyltransferase complex MTA1c in ciliates,which is composed of MTA1,MTA9,P1 and P2 proteins.Among them,MTA1 is the only component with methylation modification activity,and its deletion will result in a significant decrease in the level of 6mA modification and severe developmental defects.MTA1 has a very important biological function in ciliates,so it is of great biological significance to reveal the molecular mechanism of MTA1 catalyzed 6mA modification.In this context,the structure and substrate recognition specificity of MTA1 protein were preliminarily studied,in order to reveal its mechanism.We expressed and purified the MTA1 protein,and obtained high purity and stable MTA1 protein.We analysed the MT-A70 domain of the MTA1 protein by crystallization experiment.The crystal structure shows that the MT-A70 domain adopts a classic Rossmann fold,consisting of five β-sheets,four α-helices,and some random coils.The structure of the MT-A70 domain of MTA1 and METTL3 has a high similarity,with 37%sequence similarity,and both contain the conservative catalytic motif DPPW.In order to further study the function of MTA1 protein,we used EMSA experiment to determine the binding specificity of MTA1 to different DNA substrates.Binding experiments show that the N-terminal domain of MTA1 preferentially binds to DNA with AT motifs,while its MT-A70 domain does not have DNA binding ability.Therefore,we speculate that MTA1 methylation to modify DNA requires its N-terminal domain of DNA recognition region and C-terminal MT-A70 domain to complete together.The experimental results of this thesis preliminarily revealed the apo structure of MTA1 in ciliates DNA 6mA methyltransferase MTA1 and its DNA binding specificity,laying a foundation for further research on DNA methylation modification of 6mA.