STRUCTURAL STUDIES OF DNA RECOMBINATION AND REPAIR

DNA 重组和修复的结构研究

• 批准号: 6161973
• 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/6161973
• 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 systems. 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 are causes for various types of 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. Marty 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 tested expression of RAG proteins in E. coli. After making dozens of different fusion constructs, we have finally succeeded in making active RAG-1 in E. coli, which paves the road for both mutational studies as well as crystallographic studies of this extremely important protein. We have also cloned human RAG proteins and made constructs for expression in both E. coli and insect cells. Eventually we are going to determine the three-dimensional structures of RAG proteins 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 DNA polymerase has a proofreading mechanism to minimize the errors during replication, occasionally mismatch due to replication-errors still happens. In all living organisms there are mismatch repair systems to prevent such mutations from occurring. E. coli has a methyl-directed mismatch repair system comprising MutS, MutL and MutH proteins. The homologues of MutS and MutL proteins are also found in human. Mutations in these proteins are culprits in 90% of the hereditary nonpolyposis colorectal cancers. During last year, we have grown crystals of MutH, a sequence-specific endonuclease which is activated by MutS upon its recognition of mismatch. We have obtained two crystal forms of MutH, both diffract x-ray to atomic resolution. Recently we have solved and refined the structures of MutH, a single polypeptide chain of 229 aa, in three different crystal packing environments. We have also obtained crystals of MutL and are currently in the process of solving its structure.

脊椎动物中V（D）J基因重排是脊椎动物成熟所必需的 免疫系统。 它允许产生抗体和T细胞 受体来建立防御系统。 这种基因重排具有 在细胞发育过程中受到严格控制。错误 重排通常导致基因截短或染色体易位 这是各种类型的淋巴瘤的原因。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）， 这是启动体内基因重排的必要条件和充分条件。 博士 美国国家卫生研究院的马蒂·盖勒特的研究小组是第一个证明 RAG-1和RAG-2蛋白可以在体外启动基因重排。 小鼠RAG活性蛋白在昆虫细胞中得到过表达。 我的小组已经测试了RAG蛋白在大肠杆菌中的表达。杆菌 之后 几十种不同的融合结构，我们终于成功地 在E.大肠杆菌，这为两种突变铺平了道路。 研究以及晶体学研究， 蛋白我们还克隆了人RAG蛋白，并构建了 在E.大肠杆菌和昆虫细胞。最终我们会 确定RAG蛋白的三维结构， 与DNA识别序列的复合物， 晶体学技术 基因必须在每一个 细胞分裂周期 虽然DNA聚合酶有一个校对 最大限度地减少复制过程中的错误的机制，有时 由于复制错误导致的不匹配仍然会发生。所有活 生物体有错配修复系统来防止这种突变 防止其发生 E.大肠杆菌具有甲基指导的错配修复系统 包括MutS、MutL和MutH蛋白。MutS和MutL的同源物 蛋白质也存在于人体中。 这些蛋白质的突变是 90%的遗传性非息肉病性结直肠癌的罪魁祸首。 在去年，我们生长了MutH晶体，一种序列特异性的 核酸内切酶，其在识别 不匹配. 我们获得了MutH的两种晶型， X射线到原子分辨率。 最近，我们已经解决并完善了 MutH是一条229个氨基酸的单链多肽链， 不同的晶体包装环境。我们还获得了晶体 MutL的，目前正在解决其结构的过程。