Recombinational mechanisms of DNA repair in eukaryotes

真核生物DNA修复的重组机制

• 批准号: 6922173
• 负责人: Wolf-Dietrich Heyer
• 金额: $ 34.15万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6922173
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA repair; Saccharomyces cerevisiae; adenosinetriphosphatase; endonuclease; enzyme activity; enzyme mechanism; enzyme substrate; fungal genetics; fungal proteins; gene mutation; genetic manipulation; genetic recombination; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): Double-stranded DNA breaks (DSBs) are the primary genotoxic lesion of ionizing radiation (IR), and DSB induction appears to determine the efficacy of IR and other DMA metabolism-based anti-tumor drugs as cancer therapeutic agents.! Homologous recombination (HR) is an important DSB repair pathway, which is essential for cellular radiation resistance and genome stability. Defects in HR lead to genomic instability and have been implicated in the etiology of cancer. The long-term goal of this proposal is to elucidate the mechanism of HR with a present focus on post-synapsis and resolution, stages that are particularly poorly understood in eukaryotes. Analysis of recombination reactions reconstituted from purified proteins and in vivo experiments are combined to determine the mechanism of the dsDNA-specific ATPase Rad54 in Rad51-mediated recombination; to determine the mechanism of the mutual stimulation of the Rad51 and Rad54 proteins; and to determine the function of the Mus81-Mms4 DMA structure-specific endonuclease, which was first identified in my laboratory through its interaction with Rad54 protein. The results will provide novel mechanistic insights for Rad51, Rad54, and Mus81-Mms4, which are critical for HR in eukaryotes. The specific aims are: (1) Determine the biochemical function of Rad54 during recombination. Rad54 remodels Rad51-dsDNA complexes. We will test if Rad54 modulates access of DMA polymerases to the 3'- OH end of the invading strand in the important transition from DMA strand invasion to repair synthesis. (2) Determine the in vivo function of Rad54. Analysis of in vivo pairing intermediates in recombinational DSB repair will be used to distinguish between the 3'-OH access model and competing models. (3) Determine the mechanism of the mutual stimulation of the Rad51 and Rad54 proteins. Using separation-of-function mutants, we will establish the mechanism of the mutual stimulation of the Rad51 and Rad54 proteins and provide a rigorous test if the Rad51-Rad54 interaction is of biological significance. (4) Identify the biochemical and cellular function of the Mus81-Mms4 endonuclease. The pathways leading to resolution of junction structures in recombination are still unclear. Biochemical and in vivo experiments will determine the substrate-specificity and function of the Mus81-Mms4 endonuclease in HR.

描述（由申请人提供）：双链 DNA 断裂 (DSB) 是电离辐射 (IR) 的主要遗传毒性损伤，DSB 诱导似乎决定了 IR 和其他基于 DMA 代谢的抗肿瘤药物作为癌症治疗的功效代理.!同源重组（HR）是重要的 DSB 修复途径，对于细胞的抗辐射性和基因组稳定性至关重要。 HR 缺陷会导致基因组不稳定，并与癌症的病因学有关。该提案的长期目标是阐明 HR 机制，目前重点关注突触后和解析阶段，这些阶段在真核生物中尤其知之甚少。对纯化蛋白重组反应的分析和体内实验相结合，确定 Rad51 介导的重组中 dsDNA 特异性 ATPase Rad54 的机制；确定Rad51和Rad54蛋白相互刺激的机制；并确定 Mus81-Mms4 DMA 结构特异性核酸内切酶的功能，该酶是我的实验室通过与 Rad54 蛋白相互作用首次发现的。这些结果将为 Rad51、Rad54 和 Mus81-Mms4 提供新颖的机制见解，这对于真核生物的 HR 至关重要。具体目标是：（1）确定Rad54在重组过程中的生化功能。 Rad54 重塑 Rad51-dsDNA 复合物。我们将测试 Rad54 是否在从 DMA 链入侵到修复合成的重要转变中调节 DMA 聚合酶对入侵链 3'- OH 末端的访问。 (2)确定Rad54的体内功能。重组 DSB 修复中体内配对中间体的分析将用于区分 3'-OH 访问模型和竞争模型。 (3)确定Rad51和Rad54蛋白相互刺激的机制。利用功能分离突变体，我们将建立 Rad51 和 Rad54 蛋白相互刺激的机制，并提供严格的测试 Rad51-Rad54 相互作用是否具有生物学意义。 (4) 鉴定Mus81-Mms4核酸内切酶的生化和细胞功能。导致重组中连接结构解析的途径仍不清楚。生化和体内实验将确定 Mus81-Mms4 核酸内切酶在 HR 中的底物特异性和功能。