【作者】 邵振华；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 本论文主要研究组蛋白修饰和tRNA修饰相关蛋白的结构与功能。论文的前两章介绍了人源蛋白MCPH1串联BRCT结构域的结构与功能。MCPH1蛋白参与到DNA损伤修复过程中,它含有三个BRCT结构域,其中位于N端的第一个BRCT结构域与染色质重塑复合物SWI-SNF结合,并把SWI-SNF复合物带到DNA损伤位点,使紧密的染色质变的松散；位于C-端的第二个和第三个BRCT结构域属于串联BRCT结构域,他被证实参与结合磷酸化的组蛋白变异体H2AX (γH2AX)。γH2AX发生在DNA修复反应的早期并引发了下游的修复过程,在DNA的损伤修复过程中扮演着重要角色。在本论文中,通过采用X射线晶体学和生物化学相结合的方法,我们研究MCPH1串联BRCT结构域识别yH2AX的分子机制。首先我们用荧光偏振的方法测定了MCPH1串联BRCT与yH2AX小肽的亲和力,结果表明它们之间有很强的的相互作用。然后,我们分别解析了串联BRCT以及它与yH2AX小肽复合物的晶体结构,并与其他经典的串联BRCT结构相比较,我们的结构显示MCPH1采用一种新的RXXN motif代替RXXK motif来识别yH2AX。此外,MCPH1对磷酸化+3位的残基(Tyr,位于小肽的C末端)有选择性,它倾向于结合C末端有COOH基团的小肽,C-末端的氨基化明显减弱了它们的相互作用。我们进一步证明MCPH1串联BRCT结构域与内源性的yH2AX有相互作用。我们的研究揭示了MCPH1串联BRCT结构域识别yH2AX的分子机理。论文的后两章介绍了酿酒酵母(Saccharomyces cerevisiae) tRNA ml G9甲基转移酶scTrml0的酶活催化机制。tRNA的甲基化是一种很普遍的修饰,这种修饰对tRNA的生物学功能是必需的。然而鸟嘌呤G的N-1甲基化(m1G)在tRNA修饰中却比较罕见,目前在两个位点上发现了m1G甲基化：位于anti-codon附近的nlG37和位于D-loop和acceptor stem连接处的m1G9。据最近的文献报道,Trm10家族蛋白被发现与tRNA ml G9的形成有关,但是,该反应的催化机制以及Trm10与底物tRNA的结合机制尚不清楚。在本工作中,我们解析了酿酒酵母scTrml0与小分子底物SAH的复合物晶体结构。通过拓扑结构分析,我们发现scTrml0的酶活结构域属于SPOUT甲基转移酶家族。因为目前已知的SPOUT家族甲基转移酶都是同二聚体,所以我们使用X射线小角散射(SAXS)的方法研究了裂殖酵母spTrm10的溶液结构,结果证明spTrm10在溶液中呈现单体状态,而且它的N端区域在溶液中非常活跃。通过体外的酶活实验、ITC实验和分子docking结果,我们猜测两个保守残基参与了甲基转移反应：D210可能在酶活反应充当着转移甲基的作用,而Q118则与tRNAG9有直接的相互作用。进一步,我们用EMSA实验证实了scTrm10的N端区域和酶活结构域的碱性区域都可以识别tRNA。通过该研究,我们第一次报道了酿酒酵母scTrm10的晶体结构,并进一步分析了它催化tRNA ml G9形成的分子机制。更多还原

【Abstract】 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.更多还原