Structural And Functional Studies Of MTA1c Mediated DNA Methylation In Tetrahymena

Epigenetic modifications mainly include DNA modifications,histone modifications and non-coding RNAs,among which DNA modifications,as one of the important research elements in epigenetics,play an important role in the transcriptional regulation of genes.Among DNA modifications,the most thoroughly studied modification is 5-methylcytosine(5m C).In addition to the 5m C modification,DNA 6m A modification has received increasing attention.Recent studies have revealed that DNA 6m A is present in prokaryotic and eukaryotic genomic DNA and is involved in various biological processes,including cell cycle,nucleosome localization and mitosis.It was found that DNA 6m A is highly abundant in Tetrahymena,where methyltransferase complex MTA1 c is responsible for the 6m A modification.The MTA1 c complex contains four proteins,that are MTA1,MTA9,P1 and P2.MTA1 and MTA9 belong to the MT-A70 family and both contain the DPPW motif,but only MTA1 has methyltransferase activity.Among the complexes,both P1 and P2 can bind DNA through their SANT domains.But it is not clear how the MTA1 c complex recognizes and methylates DNA.Therefore,in this thesis,I investigated the functional and structural characteristics of the MTA1 c complex and the molecular mechanism of DNA modification of the MTA1 c complex by methylation by biochemistry,molecular biology and structural biology.To investigate the functional and structural characteristics of each protein in the MTA1 complex,I first constructed different expression vectors for MTA1,MTA9,P1 and P2 with various fusion tags,and then expressed and purified the four proteins successfully.I tried to assemble the MTA1 c complex on this basis using gel filtration chromatography,and found that MTA1 and MTA9 can form a stable binary complex,and MTA1,MTA9 and P2 could form a stable ternary complex,unfortunately,I was unsuccessful in obtaining the tetrameric complex.Then I analyzed the DNA binding ability of monomeric as well as complex proteins using EMSA and FP experiments.EMSA and FP results showed that MTA1 and P1 could weakly bind different DNA sequences,MTA9 and P2 alone had no significant DNA binding capacity.While MTA1-MTA9 binary complex proteins and MTA1-MTA9-P2 ternary complex proteins had significantly enhanced DNA binding ability.I also examined the catalytic capability of the MTA1-MTA9 complex and MTA1-MTA9-P2 complex using mass spectrometry.MS data showed that none of the DNA was methylated,I speculated that only the whole MTA1 c complex can methylate DNA 6m A.Based on the above results,I performed crystallization with different components of MTA1 complex,including MTA1-MTA9 binary complex,MTA1-MTA9-P2 ternary complex,as well as MTA1 and P1 proteins with and without different DNA.I successfully obtained the crystals of MTA1 and P1 proteins and finally resolved their structures,however,I failed to obtain the complex structure of MTA1-MTA9 and MTA1-MTA9-P2 complex.Thus,I carried out cross-linking mass spectrometry to explore the binding mode of the MTA1-MTA9 complex.The cross-linked mass spectrometry results showed that theα4 and β4-β6 of MTA1 protein bind to the binding surface formed by α4,α7,β4 and β5 of MTA9 protein,which is similar to the previously reported binding pattern of METTL3 and METTL14.In summary,in this thesis,I investigated the DNA binding ability of MTA1 c methyltransferase complex,preliminary indicated their binding mode,and obtained the crystal structures of MTA1 and P1.Although I was unable to obtain the complex structure of MTA1 c quaternary complex.this study provides an important research basis for the further study on the function of MTA1 c methyltransferase.