Structural Studies Of DNA Recombination, Repair, and Rep

DNA 重组、修复和重复的结构研究

• 批准号: 6664156
• 负责人: WEI YANG
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6664156
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; DNA replication; Escherichia coli; T cell receptor; X ray crystallography; adenosinetriphosphatase; antibody; bacterial genetics; cell cycle; crystallization; enzyme activity; gene mutation; gene rearrangement; genetic recombination; human genetic material tag; hydrolysis; intermolecular interaction; laboratory mouse; molecular cloning; nuclease; protein biosynthesis; protein structure function; tissue /cell culture

Genomic DNA has to be replicated before every cycle of cell division. Although replicative DNA polymerases have a built-in proofreading mechanism to minimize errors during replication, occasionally mismatch due to replication-error occurs. Mismatch repair systems to prevent mutations from replicative errors exist in most organisms. E. coli has a methyl-directed mismatch repair system comprising MutS, MutL and MutH proteins. Homologues of MutS and MutL proteins are also found in humans. Mutations in MutS or MutL homologs have been identified in 90% of the hereditary nonpolyposis colorectal cancers. By Oct. 2001, our group determined the crystal structures of mismatch repair proteins MutH, a conserved 40KD ATPase fragment of MutL and its complexes with nucleotides, the ATPase domain of a human MutL homologue, PMS2, alone and complexed with ATP and nonhydrolyzeable ATP analog, and the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADP/Mg2+. Last year (2001-2002), (1) based on the crystal structures, we constructed 47 MutS, MutL and MutH E. coli mutants, carried out in vitro and in vivo (in collaboration with J. Miller at UCLA) biochemical studies, and found that every mutant that fails to repair mismatch is deficient in preventing homeologous recombination. Our studies also revealed that the non-specific DNA binding property of MutL is required for mismatch repair after the DNA incision by MutH and that the structural domains in MutS work cooperatively to achieve maximal differential binding of heteroduplex versus homoduplex rather than maximal DNA binding. (2) The MutL ATPase active site shares conserved sequence motifs with DNA topoisomerases, Hsp90 and bacterial and mitochondrial kinases. Many of these proteins are potential drug targets. We found that despite the similar ATP binding pocket among these enzymes and a shared requirment of Mg ion, each of them differs in monovalent ion preference in the ATP binding pocket. Based on structure and sequence comparison between MutL and a rat mitochondrial protein kinase, we made a point mutation in MutL and converted MutL from using any monomalent ion to Na+ specifically. We have also determined the crystal structures of the wildtype and mutant MutL proteins and observed a Na+ for K+ exchange. We propose that the monovalent ion specificity may be exploited for future drug design. We have also determined the crystal structure of a hemimethylated GATC binding proten, E. coli SeqA, bound to the DNA. This project was derived from our research on how MutH recognizes hemimethylated GATC sequence and targets mismatch repair to the daughter strand specifically. The structure of SeqA-DNA complex reveals that the recognition of the methylated adenine is achieved by protein mainchain atoms via close van der Waals contacts. Most interestingly, the structure suggests a mechanism for SeqA to sequestrate DNA replication origin, oriC, from being used prematurely, repeatedly or asynchronously. A new family of DNA polymerases, the Y-family, has recently been identified. They differ from the previously known DNA polymerases in the primary sequence and in the ability of lesion-bypass and error-prone DNA synthesis. In collaboration with Dr. Roger Woodgate of NICHD, we determined the crystal structure of a Y-family DNA polymerase, Dpo4 from S. Solfotaricus, in complex with undamaged DNA and an incoming nucleotide in 2001. These crystal structures provide the first atomic view of the Y-family polymerase in action and reveal a molecular mechanism for the low fidelity DNA synthesis and bypassing modified DNA bases. Recently, we have crystallized Dpo4 in complex with damaged or mispaired DNA. The new structures suggest that Dpo4 binds an incoming nucleotide independent of correct base pairing with the template strand and shuffles the template strand to find a partner for the incoming nucleotide before catalyzing the nucleotidyl-transfer reaction. We propose tht this "free-loading" of nucleotide serves Dpo4 to bypass DNA lesions as well as to make low fidelity DNA synthesis.

基因组DNA必须在每个细胞分裂周期之前复制。虽然复制型DNA聚合酶具有内置的校对机制以最小化复制期间的错误，但偶尔会发生由于复制错误而导致的错配。在大多数生物体中存在防止复制错误突变的错配修复系统。e.大肠杆菌具有包含MutS、MutL和MutH蛋白的甲基指导的错配修复系统。MutS和MutL蛋白的同源物也在人类中发现。在90%的遗传性非息肉病性结直肠癌中已经鉴定出MutS或MutL同源物的突变。到2001年10月，我们的小组确定了错配修复蛋白MutH，MutL的保守的40KD ATP酶片段及其与核苷酸的复合物，人MutL同源物PMS 2的ATP酶结构域，单独的和与ATP和不可水解的ATP类似物的复合物，以及单独的190KD Taq MutS，与DNA的复合物，以及与DNA和ADP/Mg 2+的三元复合物的晶体结构。去年（2001 - 2002），（1）根据晶体结构，我们构建了47个MutS，MutL和MutH E。在体外和体内（与UCLA的J.米勒合作）进行了生物化学研究，发现每一个不能修复错配的突变体在防止同源重组方面都是有缺陷的。我们的研究还表明，MutL的非特异性DNA结合特性是MutH切割DNA后错配修复所必需的，MutS中的结构域协同工作，以实现异源双链体与同源双链体的最大差异结合，而不是最大DNA结合。(2)MutL ATP酶活性位点与DNA拓扑异构酶、Hsp90以及细菌和线粒体激酶共享保守序列基序。这些蛋白质中有许多是潜在的药物靶点。我们发现，尽管这些酶之间的ATP结合口袋和镁离子的共同需求相似，但它们中的每一个在ATP结合口袋中的单价离子偏好不同。基于MutL与大鼠线粒体蛋白激酶的结构和序列比较，我们对MutL进行了点突变，将MutL从使用任何单价离子转换为Na+特异性。我们还确定了野生型和突变MutL蛋白的晶体结构，并观察到Na+交换K+。我们建议可以利用单价离子的特异性进行未来的药物设计。 我们还测定了半甲基化GATC结合蛋白E. coliSeqA，与DNA结合。本项目来源于我们对MutH如何识别半甲基化GATC序列并特异性靶向子链错配修复的研究。SeqA-DNA复合物的结构表明，甲基化腺嘌呤的识别是通过蛋白质主链原子通过紧密的货车德瓦尔斯接触实现的。最有趣的是，该结构表明SeqA可以隔离DNA复制起点oriC，防止其过早、重复或异步使用。 最近发现了一个新的DNA聚合酶家族，Y家族。它们与先前已知的DNA聚合酶在一级序列以及病变旁路和易错DNA合成能力方面不同。与NICHD的Roger Woodgate博士合作，我们确定了Y家族DNA聚合酶Dpo 4的晶体结构。2001年，Solfotaricus与未受损的DNA和进入的核苷酸复合。这些晶体结构提供了Y-家族聚合酶作用的第一个原子视图，并揭示了低保真度DNA合成和绕过修饰的DNA碱基的分子机制。最近，我们已经使Dpo4与受损或错配的DNA复合结晶。新的结构表明，Dpo4结合一个传入的核苷酸独立于正确的碱基配对与模板链和洗牌的模板链，以找到一个合作伙伴的传入的核苷酸催化核苷酸转移反应之前。我们认为这种"自由装载"的核苷酸有助于Dpo 4绕过DNA损伤以及进行低保真度DNA合成。