Structural Studies Of DNA Recombination And Repair

DNA重组与修复的结构研究

• 批准号: 6532120
• 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/6532120
• 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 polymerase has a built-in proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication-errors still happens. Mismatch repair systems to prevent such mutations from occurring 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 human. Mutations in these proteins are identified in 90% of the hereditary nonpolyposis colorectal cancers. In the three years before Oct. 2000, our group determined the crystal structures of mismatch repair proteins MutH, a conserved 40KD ATPase fragment of MutL and its complexes with nucleotides, and the 190 Kd Taq MutS alone, complexed with DNA, and as a ternary complex with DNA and ADP/Mg2+. Last year (2000-2001), (1) we carried out mutagenesis and biochemical studies of E. coli MutS and based on our results proposed an ATP-assisted mismatch recognition proofreading mechanism, which is similar to the proofreading in protein synthesis, (2) we determined the crystal structure of the ATPase fragment of Pms2, a human homolog of MutL and characterized its unusual monomeric ATPase activity, (3) in collaboration with Dr. Thomas Kunkel, we initiated the studies of asymmetric DNA binding and ATPase hydrolysis in heterodimeric eukaryotic MutS homologs and characterized the residues essential for mismatch recognition versus residues required for general protein-DNA interactions, (4) we generated 47 functional mutations in E. coli MutS, MutL and MutH based on our crystal structures and in collaboration with Dr. Jeffrey Miller of UCLA, we studied their in vivo mismatch repair and DNA recombination phenotypes. Combined with our in vitro studies, we have produced a comprehensive profile of the function and mechanism of these mismatch repair proteins. A new family of DNA polymerases, the Y-family, has recently been identified. They differ from the previously known DNA polymerase in the primary sequence and in lesion-bypass and error-prone DNA synthesis. In collaboration with Dr. Roger Woodgate of NICHD, we have determined the crystal structure of a Y-family DNA polymerase, Dpo4 from S. Solfotaricus, in complex with the substrate DNA and an incoming nucleotide. The crystal structures provide the first view of the Y-family polymerase in action and reveal the molecular mechanism for low fidelity DNA synthesis and bypassing modified DNA bases. We have continued our research of V(D)J recombination. V(D)J gene rearrangement in vertebrates is essential for the maturation of immune systems, which allows the generation of antibodies and T-cell receptors to build up the defense system. V(D)J gene rearrangement is a type of site-specific DNA recombination. Two proteins, RAG-1 and RAG-2 (recombination activation gene products), are necessary and sufficient to turn on the gene rearrangement in vivo, but they are difficult to obtain in a pure and soluble form. We have cloned and produced several soluble fragments of the mouse RAG1 proteins. Even though soluble, pure and in ample supply, these fragments still resist our crystallization attempts. Interestingly, one of the fragments contains a nuclease activity, which might have cellular functions. In collaboration with Dr. Marjorie Oettinger of Massachusetts General Hospital, we are characterizing the nuclease activity.

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过程中的错误，但偶尔仍会发生由于复制错误而导致的错配。大多数生物中都存在防止这种突变发生的错配修复系统。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。这些蛋白质的突变在90%的遗传性非息肉病性结直肠癌中被鉴定。在2000年10月之前的三年中，我们研究组测定了错配修复蛋白MutH、MutL的保守的40KD ATP酶片段及其与核苷酸的复合物、190KD Taq MutS单独、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（2000 - 2001年），（1）我们进行了E. coliMutS中，提出了一种类似于蛋白质合成中的错配识别校正机制，（2）确定了MutL的同源物Pms 2的ATP酶片段的晶体结构，并表征了其不寻常的单体ATP酶活性。（3）与托马斯·昆克尔博士合作，我们在异源二聚体真核MutS同源物中启动了不对称DNA结合和ATP酶水解的研究，并表征了错配识别所必需的残基与一般蛋白质-DNA相互作用所需的残基。在我们的晶体结构的基础上，我们与UCLA的Jeffrey米勒博士合作，研究了它们的体内错配修复和DNA重组表型。结合我们的体外研究，我们已经产生了这些错配修复蛋白的功能和机制的全面概况。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、病变旁路和易错DNA合成方面不同。与NICHD的Roger Woodgate博士合作，我们已经确定了Y家族DNA聚合酶Dpo 4的晶体结构。Solfotaricus，与底物DNA和进入的核苷酸复合。晶体结构提供了Y家族聚合酶作用的第一个视图，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。我们继续对V（D）J复合进行了研究。脊椎动物中的V（D）J基因重排对于免疫系统的成熟是必不可少的，其允许产生抗体和T细胞受体以建立防御系统。V（D）J基因重排是一种位点特异性DNA重组。两种蛋白质，RAG-1和RAG-2（重组激活基因产物），是必要的，足以在体内启动基因重排，但它们很难获得纯的和可溶的形式。我们已经克隆并产生了小鼠RAG1蛋白的几个可溶性片段。尽管这些碎片是可溶的，纯净的，而且供应充足，但它们仍然抵制我们的结晶尝试。有趣的是，其中一个片段含有核酸酶活性，可能具有细胞功能。我们与马萨诸塞州总医院的Marjorie Oettinger博士合作，对核酸酶活性进行了表征。