STRUCTURAL STUDIES OF DNA RECOMBINATION AND REPAIR

DNA 重组和修复的结构研究

• 批准号: 2439086
• 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/2439086
• 关键词: DNA directed DNA polymerase; DNA repair; Insecta; X ray crystallography; animal genetic material tag; gene mutation; gene rearrangement; genetic recombination; immunoglobulin genes; laboratory mouse; molecular cloning; nucleic acid sequence; protein structure function; tissue /cell culture; transcription factor

V(D)J gene rearrangement in vertebrates is essential for the maturation of immune system. It allow the generation of antibodies and T-cell receptors to build up the defense system. Such gene rearrangement has to be tightly controlled during cell development. Erroneous rearrangement often leads to gene truncation or chromosome translocation which often causes various lymphomas. 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. Dr. Martin Gellert's group here at NIH is the first to demonstrate purified RAG-1 and RAG-2 proteins can initiate gene rearrangement in vitro. Active RAG proteins from mouse have been over-expressed in insect cells. My group has scaled up the protein production. We are currently characterizing both RAG-1 and RAG-2 proteins biochemically and plan to determine the three-dimensional structures of each protein and their complexes with the DNA recognition sequences using x-ray crystallographic techniques. Genes have to be replicated before every cycle of cell division. Although DAN polymerase has a proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication- errors still happens. In all living organisms there is such a mismatch repair system to prevent mutation from occurring. Dr. Hsieh's group at NIDDK has cloned the mismatch-repair protein MutS and MutL from thermus aquaticus (TAQ) and overexpressed the proteins in E.coli. TAQ proteins are homologues of E.coli proteins and recognize mismatch or 1-3 nucleotide-loopout in DNA duplexes. The homologues of these mismatch-repair proteins are also found in human. Mutations in these proteins are found in 90% of the hereditary nonpolyposis colorectal cancer. We are currently crystallizing TAQ MutS protein and its complexes with abnormal DNAs. Once diffracting crystals are obtained, we will determine the atomic structure by x-ray diffraction method and try to understand the mechanism of mismatch DNA repair.

脊椎动物中V（D）J基因重排是脊椎动物发生 免疫系统的成熟。 它可以产生抗体 和T细胞受体来建立防御系统。 这样的基因 在细胞发育过程中必须严格控制重排。 错误的重排往往导致基因截短或染色体 易位，这往往会导致各种淋巴瘤。 V（D）J基因 重排是一种位点特异性DNA重组。 两 蛋白质，RAG-1和RAG-2（重组激活基因产物）， 是开启基因重排的必要条件， vivo. 美国国立卫生研究院的马丁·盖勒特博士的团队是第一个 证明纯化的RAG-1和RAG-2蛋白可以启动基因表达， 体外重排。 来自小鼠的活性RAG蛋白已经被 在昆虫细胞中过度表达。 我的团队已经将蛋白质 生产 我们目前正在表征RAG-1和RAG-2 蛋白质，并计划确定三维 每种蛋白质及其与DNA的复合物的结构 使用X射线晶体学技术识别序列。 基因必须在每个细胞分裂周期之前复制。 尽管DAN聚合酶具有校正机制以最小化聚合酶的活性， 复制过程中的错误，偶尔会由于复制而不匹配- 错误仍然会发生。 在所有生物体中， 错配修复系统，以防止突变的发生。 博士 NIDDK的Hsieh小组克隆了错配修复蛋白MutS， MutL从Thermus aquaticus（TAQ）和过表达的蛋白质， E.coli. TAQ蛋白是大肠杆菌蛋白的同源物， 错配或DNA双链体中的1-3个核苷酸环出。 的同系物 这些错配修复蛋白也存在于人类中。 突变 这些蛋白质存在于90%的遗传性非息肉病 结肠直肠癌 我们目前正在结晶TAQ MutS蛋白 以及它与异常DNA的复合物 一旦衍射晶体 得到的，我们将确定原子结构的x射线衍射 方法，并试图了解错配DNA修复的机制。