Structure And Function Of Proteins Involved In Phosphorylated Histone And Methylated TRNA

The thesis focus on structure and function of proteins that are involved in histone modification and tRNA modification.In the first two chapters, we introduce the structure and function of human protein MCPH1tandem BRCT domains, which is involved in DNA damage response pathway. MCPH1contains three BRCT domains, one is at N-terminus and the tandem BRCT are at C-terminus. The N-terminal BRCT can directly bind to chromatin remodeling complex SWI-SNF and bring the complex to DNA damage sites, then the SWI-SNF complex can relax compact chromatin; the C-terminal BRCT2-BRCT3(tandem BRCT domains) are involved in interacting with yH2AX in DNA damage response. yH2AX is an early event in DNA response and triggers downstream pathway. In addition, yH2AX plays an important role in the DNA response pathway. In our study, we carried out X-ray crystallography and biochemical methods to investigate the molecular mechanism MCPH1tandem BRCT domains recognize yH2AX,. First of all, we determined the binding affinity between MCPH1with yH2AX peptide using fluorescence polarization (FP) assays, the results revealed that the binding affinity is very strong, which is consistent with previous study. Then we solved the crystal structures of MCPH1tandem BRCT alone as well as in complex with yH2AX peptide. Compared with other structures of tandem BRCT domains, MCPH1uses a novel motif (RXXN) to recognize yH2AX instead of RXXK motif in other BRCT domains. Additionally, MCPH1tandem BRCT domains show a binding selectivity on pSer+3(Tyr) and prefer to bind phosphopeptide with free COOH-terminus. The amination of C-terminus in peptide reduced the binding affinity. Taken together, our research provided the molecular mechanism of yH2AX recognition by MCPHl tandem BRCT domain.In the last two chapters, we talked about structure and function of tRNA ml G9methyltransferase scTrm10from Saccharomyces cerevisiae. The methylation of tRNA is very common modification, it is very necessary for tRNA function. However, the m1G modification is very rare and has been identified in tRNA only in two positions until now:one is m1G37modification, which is present at Position37(adjacent to3’ of the anticodon) of the tRNA; the other is m1G9modification, which is present at Position9and between D-loop and acceptor stem junction. The m1G9modification was recently found to be catalyzed by Trm10family of methyltransferases, but the molecular mechanism and substrate tRNA binding mechanism of Trm10is unclear. In this thesis, we solved the crystal structure of Saccharomyces cerevisiae scTrm10in complex with cofactor SAH. From its topology structure, we can see that TrmlO belongs to SPOUT family. SPOUT family methyltransferases studied to date are known to functionally exist as homodimers, so we performed small angle X-ray scattering (SAXS) to study the solution structure of spTrmlO from Schizosaccharomyces pombe. The SAXS analysis results revealed that spTrmlO functions as a monomer in solution and its N-terminal extension is very flexible. Additionally, using in vitro tRNA methyltransferase activity, ITC experiments and molecular docking, we suggested that two residues could be directly involved in methyl group transferring reaction:D210could play an important role in methyl group transferring, Q118may be involved in substrate G9binding. Furthermore, our EMS A results showed that N-terminal extension and basic surface on the TrmlO methyltransferase domain are all essential to bind the substrate tRNA. In all, we reported the first crystal structure of scTrm10family, and our work provides an insight into the molecular mechanism that how Trm10family catalyzes tRNA mlG9formation.