S CEREVISIAE RAD54P IN HOMOLOGOUS DNA RECOMBINATION

RAD52上位组负责通过同源重组修复酿酒酵母中的大部分双链DNA断裂。该组至少包含9个基因：RAD50、RAD51、RAD52、RAD54、RAD55、RAD57、RAD59、Mre11和XRS2(1，2)。这些基因中的许多突变导致对DNA损伤剂的敏感性增加，如电离辐射或甲基甲烷磺酸盐，这些物质会产生双链DNA断裂(3，4)。此外，当细胞经过减数分裂时，含有该上位组许多成员突变的菌株是不能存活的，而减数分裂需要同源重组(4，5)。虽然在阐明T4噬菌体和大肠杆菌中同源重组的机制步骤和确定同源重组的酶方面已经取得了相当大的进展，但我们目前对真核DNA重组的理解水平仍然有限。这项建议的重点是扩大我们对酿酒酵母这一过程的了解。目前，Kovalczykowski等人正在研究Rad51蛋白、Rad52蛋白和RPA的作用。我的目标是用生化方法来确定Rad54蛋白在同源重组中的功能。鉴于到目前为止的研究，我认为Rad54蛋白可能作为DNA染色质重塑因子，或作为一种能够刺激同源重组的分支迁移阶段的酶。我计划使用噬菌体T4和大肠杆菌重组系统来检测Rad54蛋白的酶特性，以帮助指导最初的研究。

The RAD52 epistasis group is responsible for repairing the majority of double-stranded DNA breaks via homologous recombination in S. cerevisiae. This group contains at least nine genes: RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2 (1,2). Mutations in many of these genes result in an increased sensitivity to DNA damaging agents, such as ionizing radiation or methylmethanesulfonate, that produce double stranded DNA breaks (3,4). In addition, strains containing mutations in many members of this epistasis group are inviable when the cells go through meiosis, where homologous recombination is required (4,5). While considerable progress has been made toward elucidating the mechanistic steps of and determining the enzymes responsible for homologous recombination in bacteriophage T4 and in E. coli, our current level of understanding of eukaryotic DNA recombination remains limited. The focus of this proposal is to expand our knowledge of this process in S. cerevisiae. Currently, the roles of Rad5l protein, Rad52 protein and RPA are being investigated by the Kowalczykowski group and others. My goal is to use biochemical methods to define the function of the Rad54 protein in homologous recombination. Given the studies to date, I believe that Rad54 protein may have a role either as a DNA chromatin remodeling factor, or as an enzyme capable of stimulating the branch migration phase of homologous recombination. I plan to examine the enzymatic characteristics of Rad54 protein using the bacteriophage T4 and E. coli recombination systems to help guide the initial studies.