End Joining Reaction in DNA Repair & V(D)J Recombination

DNA 修复中的末端连接反应

• 批准号: 6615076
• 负责人: GILBERT CHU
• 金额: $ 26.15万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615076
• 关键词: DNA binding protein; DNA damage; DNA repair; cell line; enzyme activity; enzyme inhibitors; gene mutation; genetic recombination; phosphorylation; protein kinase; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Ionizing radiation and V(D)J recombination generate DNA double-strand breaks (DSBs), which are repaired by non-homologous end-joining (NHEJ). Proteins required for NHEJ include DNA-dependent protein kinase (DNA-PK), which consists of a DNA binding subunit (Ku) and a catalytic subunit (DNA-PKcs). It is not yet known how DNA-PK and other factors bring the broken ends of the DNA together, process the ends for joining, and regulate the reaction by phosphorylation. The long-term objective is to elucidate each step in NHEJ, which will lead to a better understanding of cancer, immunity, and aging. The specific aims are:1.To define the mechanism for synapsis of DNA ends during NHEJ. Preliminary studies show that DNA-PKcs brings DNA ends together in a synaptic complex at low salt. Proposed studies will study the effect of DNAPKcs autophosphorylation on DNA synapsis, determine the factors required for synapsis to occur at physiological salt, and determine the number of DNA-PKcs molecules in the synaptic complex.2. To define how the DNA ends are processed for joining during NHEJ. A cell-free system for NHEJ detected efficient end-processing in wild type extracts and deficient end-processing in extracts from Werner's Syndrome, a disease of premature aging and cancer, and Nijmegan Breakage Syndrome, a disease of radiation sensitivity and cancer. Experiments will define the roles of the associated proteins, Wm and Nbs1. End-processing included templated nucleotide addition and increased with addition of dNTPs. Experiments will determine if DNA polymerase(s) are involved. The cell free system will be used to characterize the joining of hairpin ends, which occurs during V(D)J recombination, identify the endonuclease that opens the hairpin ends, and determine how hairpin opening is regulated by DNA-PKcs.3. To define how NHEJ is regulated by protein phosphorylation. To determine the role of DNA-PKcs kinase activity in NHEJ, DNA-PKcs-mutant extracts will be supplemented with the active kinase or wortmannin-inactivated kinase and assayed for processing and joining of DNA ends. Experiments will determine whether phosphatases play a role in NHEJ, and identify physiologically relevant targets of DNA-PKcs.

描述(申请人提供)：电离辐射和V(D)J重组产生DNA双链断裂(DSB)，通过非同源末端连接(NHEJ)修复。NHEJ所需的蛋白质包括DNA依赖蛋白激酶(DNA-PK)，它由DNA结合亚基(Ku)和催化亚基(DNA-PKcs)组成。目前尚不清楚DNA-PK和其他因子是如何将DNA的断端连接在一起，处理末端以供连接，并通过磷酸化来调节反应的。长期目标是阐明NHEJ的每个步骤，这将导致对癌症、免疫和衰老的更好理解。具体目的是：1.明确NHEJ过程中DNA末端突触的机制。初步研究表明，DNA-PKcs在低盐条件下将DNA末端聚集在一个突触复合体中。建议的研究将研究DNAPKcs自动磷酸化对DNA突触的影响，确定在生理盐条件下发生突触所需的因素，并确定突触复合体中DNA-PKcs分子的数量。以确定在NHEJ期间如何处理DNA末端以供连接。NHEJ的无细胞系统检测到野生型提取物中的有效末端加工，而沃纳综合征(一种早衰和癌症疾病)和奈梅根断裂综合征(一种辐射敏感性疾病和癌症)提取物中的末端加工不足。实验将确定相关蛋白质Wm和Nbs1的作用。末端加工包括模板核苷酸加成，并随着dNTPs的加入而增加。实验将确定是否与DNA聚合酶(S)有关。该无细胞系统将用于表征V(D)J重组过程中发夹末端的连接，鉴定打开发夹末端的内切酶，并确定DNA-PKcs如何调节发夹末端的打开。明确NHEJ是如何通过蛋白质磷酸化来调节的。为了确定DNA-PKcs激酶活性在NHEJ中的作用，DNA-PKcs突变提取物将添加活性激酶或Wortmannin失活激酶，并进行DNA末端的加工和连接分析。实验将确定磷酸酶是否在NHEJ中发挥作用，并确定DNA-PKcs的生理相关靶点。