MECHANISMS OF DOUBLE STRAND BREAK REPAIR IN YEAST

拟议研究的目的是确定 酵母DNA修复蛋白Rad55和Rad57。这两种蛋白质是 最近发现的RecA同源物表明在DNA中有直接作用 修复和重组。RAD55和RAD57基因是 修复双链断裂，用于自发的有丝分裂重组和 用于减数分裂。缺乏双链断裂所需的功能 修复有有害的影响，如增加诱变率和 染色体丢失和减数分裂致死。就像癌症易感性一直以来 与DNA修复功能的缺陷有关，有可能 哺乳动物中双链断裂修复途径的缺陷也将 参与癌症的进展。此外，由于RAD基因是 在酵母中进行基因打靶所需的，了解它们在 酵母可能导致改进的基因策略的发展 以哺乳动物细胞为靶点。这项提案的重点是确定 RAD55和RAD57的生化功能及其相互作用 其他Rad蛋白，特别是RAD51。RAD55和RAD57纯化自 酵母表现出ATP刺激的与单链DNA的结合，但没有 与双链DNA显著结合。将对纯化的蛋白质进行测试 在RecA样活性的其他生化分析中，包括 互补单链DNA、链交换、D-环的再退火 环状分子的形成、配对和Holliday的分支迁移 交汇点。由于rad55和rad57突变体具有相似的表型，并且 蛋白质通过两个杂交系统相互作用，两个纯化的蛋白质将 混合在一起以确定复合体是改变了还是增加了 与单个蛋白质的活性进行比较。这两种蛋白质将 也可以在RAD51的联合反应中进行测试，以确定它们是否 作为RAD51介导链交换的刺激因子 反应。Rad55和Rad57与其他Rad之间的复杂关系 蛋白质，或新型蛋白质，将用亲和力来确定 层析、免疫沉淀和凝胶过滤层析。 我们还计划研究RAD基因在质粒缺口修复中的作用。 实验旨在验证我们的假设，即RAD51途径 参与DNA修复事件，这些事件被解决为非交叉。这个 供体序列定位对GAP的影响 还将研究RAD品系的修复情况。最后，我们计划 筛选抑制DNA修复的RAD51高活性等位基因 Rad55/57突变体的缺陷。这些等位基因的特征可以 提供对这些蛋白质功能的洞察。

The purpose of the proposed research is to determine the functions of the yeast DNA repair proteins Rad55 and Rad57. These two proteins were recently identified as RecA homologues suggesting a direct role in DNA repair and recombination. The RAD55 and RAD57 genes are required for the repair of double-strand breaks, for spontaneous mitotic recombination and for meiosis. The lack of functions required for double-strand break repair has deleterious effects, such as elevated rates of mutagenesis and chromosome loss, and meiotic lethality. As cancer susceptibility has been associated with defects in DNA repair functions it is possible that defects in the double-strand break repair pathway in mammals will also be involved in cancer progression. Furthermore, as the RAD genes are required for gene targeting in yeast, an understanding of their role in yeast may lead to the development of improved strategies for gene targeting in mammalian cells. The focus of this proposal it to determine the biochemical functions of Rad55 and Rad57 and their interactions with other Rad proteins in particular Rad51. Rad55 and Rad57 purified from yeast exhibit ATP-stimulated binding to single-stranded DNA, but no significant binding to duplex DNA. The purified proteins will be tested in other biochemical assays for RecA-like activities, including reannealing of complementary single-stranded DNA, strand exchange, D-loop formation, pairing of circular molecules and branch migration of Holliday junctions. As rad55 and rad57 mutants have similar phenotypes, and the proteins interact by the two hybrid system the two purified proteins will be mixed to determine whether the complex has altered or increased activity compared with the individual proteins. The two proteins will also be tested in combined reactions with Rad51 to determine whether they act as stimulatory factors for the Rad51-mediated strand-exchange reaction. Complex formation between Rad55 and Rad57 with other Rad proteins, or novel proteins, will be determined using affinity chromatography, immunoprecipitation and gel filtration chromatography. We also plan to investigate the role of RAD genes in plasmid gap repair. Experiments are designed to test our hypothesis that the Rad51 pathway is involved in DNA repair events that are resolved as non-crossovers. The effect of donor sequence location either plasmid or chromosomal on gap repair in rad strains will also be investigated. Finally, we plan to screen for hyperactive alleles of RAD51 that suppress the DNA repair defect of rad55/57 mutants. Characterization of such alleles may provide insight into the function of these proteins.