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）的主要遗传毒性病变（IR），而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剂的疗效。同源重组（HR）是重要的DSB修复途径，这对于细胞辐射抗性和基因组稳定性至关重要。人力资源缺陷导致基因组不稳定性，并与癌症的病因有关。该提案的长期目标是阐明人力资源机制，目前关注于后同伴和解决方案，这是真核生物中特别鲜为人知的阶段。从纯化的蛋白质和体内实验中重新组建的重组反应分析，以确定在RAD51介导的重组中DsDNA特异性ATPase RAD54的机理；确定Rad51和Rad54蛋白的相互刺激的机制；并确定MUS81-MMS4 DMA结构特异性核酸内切酶的功能，该核酸酶特异性核酸酶首次通过与RAD54蛋白的相互作用在我的实验室中鉴定出来。结果将为RAD51，RAD54和MUS81-MMS4提供新的机械见解，这对于真核生物中的HR至关重要。具体目的是：（1）确定重组过程中RAD54的生化功能。 RAD54重塑RAD51-DSDNA复合物。我们将测试RAD54是否调节了从DMA链入侵到修复合成的重要过渡中DMA聚合酶进入入侵链的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。使用功能分离突变体，我们将建立RAD51和RAD54蛋白相互刺激的机制，如果Rad51-Rad54相互作用具有生物学意义，则可以提供严格的测试。 （4）确定MUS81-MMS4核酸内切酶的生化和细胞功能。导致分辨出重组中连接结构的途径尚不清楚。生化和体内实验将确定HR中MUS81-MMS4核酸内切酶的底物特异性和功能。