RECOMBINATIONAL MECHANISMS OF DNA REPAIR IN EUKARYOTES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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DNA代谢的抗肿瘤药物作为癌症治疗剂的疗效。同源重组（HR）是一种重要的DSB修复途径，对细胞的辐射抗性和基因组稳定性至关重要。HR缺陷导致基因组不稳定性，并与癌症的病因学有关。这项建议的长期目标是阐明HR的机制，目前的重点是突触后和分辨率，这些阶段在真核生物中特别缺乏了解。结合对纯化蛋白重组反应的分析和体内实验，以确定双链DNA特异性ATP酶Rad54在Rad51介导的重组中的机制，确定Rad51和Rad54蛋白相互刺激的机制，并确定Mus81-Mms4 DNA结构选择性内切酶的功能，它是在我的实验室通过与Rad54蛋白的相互作用首次被发现的。这些结果将为Rad51，Rad54和Mus81-Mms4提供新的机制见解，这对真核生物中的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是否在从DNA链侵入到修复合成的重要转变中调节DNA聚合酶对侵入链的3'-OH末端的接近。 (2)确定Rad54的体内功能。重组DSB修复中的体内配对中间体的分析将用于区分3'-OH进入模型和竞争模型。(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，并提供一个严格的测试，如果Rad51-Rad54相互作用具有生物学意义。(4)确定Mus81-Mms4内切核酸酶的生物化学和细胞功能。导致在重组中解析结结构的途径仍然不清楚。生物化学和体内实验将确定底物特异性和Mus81-Mms4内切核酸酶在HR中的功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 DNA 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 DNA structure-selective 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 DNA polymerases to the 3'-OH end of the invading strand in the important transition from DNA 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.