Structural And Functional Research Of Human DNA Damage Response Protein PTIP And TRNA Methyltransferase Trm10from Schizosaccharomyces Pombe

The first two chapters in my thesis is about the structural and functional research of human PTIP, which is involved in the DNA damage response (DDR) pathway. DDR pathyway is a basic cellular process which is indispensable for maintaining genome stability. Moreover, the process is vitally important for cells and organisms function. Once DNA was damaged by endogenous or exogenous factors during different phases of cell cycle, many proteins can be recruited to DNA lesion sites, where these proteins can compose a complex network to activate DNA repair mechanisms. In mammalian cells, histone variant H2AX C-terminal phosphorylation is one of the first chromatin modifications and is an important early event in DDR pathway, which occurs for the purpose of recruiting repair proteins to the DNA double-stranded breaks sites (DSBs). Particularly, the tandemBRCT (BRCA1C-terminal domain)-containing proteins, such as hMDCl, hMCPH1, spBrc1and spCrb2(53BP1homologue in Schizosaccharomyces pombe), can bind to phosphor-H2AX (γH2AX) or phosphor-H2A (γH2A, in yeast).We take protein PTIP as an object. PTIP contains six BRCT domains:two at the amino terminus and the other four at the carboxyl terminus. Previous studies indicated that the second BRCT domain of PTIP can directly interact with PA1, and the four C-terminal BRCT domains are needed for the foci formation with γH2AX after IR (ionizing radiation).Previous studies indicated that, by the oriented peptide library screen, PTIP BRCT5-BRCT6domains have been proved to be sufficient for binding with a phospho-peptide pS/T-Q-V-F motif. Since human γH2AX C-terminal tail has a similar motif (pS-Q) corresponding to the optimal BRCT5-BRCT6binding sequence. Moreover, the C-terminal tandem BRCT domains of Brcl (PTIP homologue in S. pombe) can bind to γH2A, it implies that PTIP could interact with γH2AX. The results of fluorescence polarization assays (FPA) demonstrated the high binding ability between BRCT5-BRCT6and γH2AX peptide in vitro, and we further solved the crystal structure of BRCT5-BRCT6-γH2AX tail complex at2.15A resolution. Our structureindicated that PTIP BRCT5-BRCT6has the conserved residues that can bind to phopho-substrates and has a selectivity at pSer+3position. In all, our results provided the molecular basis of γH2AX recognition by human PTIP BRCT5-BRCT6.The last two chapters is about the structural and functional research of tRNA m1G9methyltransferase Trm10from Schizosaccharomyces pomhe. Transfer RNA (tRNA) methylation is necessary for the proper biological function of tRNA, which not only effect the efficiency of protein translation, but also effect tRNA3D proper fold. My thesis focus on tRNA m1G methylation. However, thus far, only two methylated positions in tRNA, one is m1G37methyltaion, which is catalyzed by TrmD in bacteria and Trm5in archaea and eukaryotes. Trm5belong to classic RFM superfamily and TrmD belong to SPOUT superfamily. Another is the N1methylation of guanine at Position9(m1G9) of tRNA, which is widely identified in eukaryotes and archaea, was found to be catalyzed by the Trm10family of methyltransferases (MTases).There is no identifiable sequence homo logy between the spTrmlO family and previously characterized MTases,including m1G MTases, such as Trm5and TrmD, to investigate the catalytic mechanism of Trm10family. Here, we report the first crystal structures of the tRNA MTase spTrmlO from Schizosaccharomyces pombe in the presence and absence of its methyl donor product S-adenosyl-homocysteine (SAH). At the same time, my collaborator demonstrated its ortholog scTrm10from Saccharomyces cerevisiae in complex with SAH. Our crystal structures indicated that the MTase domain (the catalytic domain) of the spTrm10displays a typical SpoU-TrmD (SPOUT) fold. Furthermore, small angle X-ray scattering analysis reveals that Trm10behaves as a monomer in solution, whereas other members of the SPOUT superfamily all function as homodimers. We also performed tRNA MTase assays, isothermal titration calorimetry (ITC) experiments, electrophoretic mobility shift assays (EMSA) and mutational analysis to provide insights into the substrate tRNA recognition mechanism of Trm10family MTases.