RECOMBINATIONAL MECHANISMS OF DNA REPAIR IN EUKARYOTES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 双链DNA断裂(DSB)是电离辐射(IR)的主要遗传毒性损伤，DSB的诱导似乎决定了IR和其他基于DNA代谢的抗肿瘤药物作为癌症治疗药物的疗效。同源重组(HR)是一条重要的DSB修复途径，对细胞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，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解，这两个阶段在真核生物中尤其鲜为人知。通过对纯化蛋白重组反应的分析和体内实验相结合，确定了dsDNA特异性ATPase Rad54在RAD51介导的重组中的作用机制，确定了RAD51和Rad54蛋白相互刺激的机制，并确定了MUS81-Mms4 DNA结构选择内切酶的功能，该酶是本实验室首次通过与Rad54蛋白的相互作用而鉴定的。这些结果将为RAD51、RAD54和MUS81-MMS4提供新的机制见解，这些都是真核生物中对HR至关重要的。 具体目的是：(1)测定重组过程中RAD54的生化功能。RAD54重塑RAD51-dsDNA复合体。我们将测试在从DNA链入侵到修复合成的重要转变中，Rad54是否调节DNA聚合酶对侵袭链3‘-OH端的访问。 (2)测定RAD54的体内功能。对重组DSB修复中体内配对中间产物的分析将用于区分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)对MUS81-MMS4内切酶的生化和细胞功能进行鉴定。在重组中导致连接结构分解的途径仍然不清楚。生化和体内实验将确定MUS81-MMS4内切酶在HR中的底物特异性和功能。

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.