ANALYSIS OF YEAST DNA REPAIR GENES RAD51, 52, AND 54

越来越多的证据表明，重组修复在人类细胞中很重要，并可能在预防癌症方面发挥作用。酵母和人类的RAD51基因编码链转移蛋白，参与重组和DNA修复。在。在酿酒葡萄球菌中，Rad51家族的相关蛋白还包括Rad55和Rad57，它们与Rad51形成异源二聚体相互作用。在人类细胞中，该家族中的6个蛋白（HsRad51、XRCC2、XRCC3、Rad51B/hRec2、Rad51C和Rad51D）可能参与重组修复和维持基因组稳定性。我们确定了所有这些蛋白质之间的特定相互作用。这些相互作用表明，人类蛋白质要么是重组体的一部分，要么形成异源二聚体和三聚体，在一个或多个修复途径中起作用。一种假说认为，人类Rad51相关蛋白，如酵母Rad55和Rad57，在DNA修复中通过与Rad51物理合作发挥作用。这一假设将通过确定这些蛋白质如何相互作用来验证，这些信息将为我们了解它们的功能提供重要线索。我们的目标是更严格地检查这些相互作用，并确定它们的生物学意义。提出了共同免疫沉淀（co-IP）和细胞学定位实验来扩展我们的酵母双杂交结果。我们将对DNA损伤诱导的HsRad51病灶进行表征，看它们是否含有其他rad51相关蛋白。凝胶过滤研究将确定这些是一个大的复杂或不同的杂多聚体。改性酵母双杂交实验将确定哪些对蛋白质相互作用可以同时发生，哪些可以竞争或合作发生。双杂交结果的一个主要优势是，它们可以迅速提出模型，然后通过协同ip、凝胶过滤和免疫定位实验进行测试。综合数据将告诉我们这些蛋白质是否形成重组体，如果是，将阐明其结构。双杂交缺失分析将用于绘制XRCC2、XRCC2和Rad51C的结合域，分离出相互作用缺陷突变。这些相互作用缺陷突变将被测试，看看它们是否可以补充敲除突变和/或它们是否可以作为显性负等位基因。在双杂化体系中发现了Rad51C与ikk - β的相互作用。ikk - β是一种参与NF-kappaB活化的激酶，是对x射线和其他刺激作出反应的系统的一部分。我们将尝试从生化角度证实观察到的rab51c - ikk - β相互作用，并确定Rad51C是否被磷酸化。如果是这样，我们将测试磷酸化是否在x射线或MMC处理下发生变化，并通过分析关键残基上可能没有磷酸化的突变体Rad51C来检验其生物学意义。

There is increasing evidence that recombinatorial repair is important in human cells, and likely plays a role in preventing cancer. The yeast and human RAD51 genes encode strand-transfer proteins involved in recombination and DNA repair. In. S. cerevisiae, the Rad51 family of related proteins also includes Rad55 and Rad57, which form a heterodimer that interacts with Rad51. In human cells, six proteins in this family (HsRad51, XRCC2, XRCC3, Rad51B/hRec2, Rad51C and Rad51D) likely participate in recombinatorial repair and in maintaining genomic stability. We identified specific interactions between all of these proteins. These interactions suggest that the human proteins are either part of a recombinosome, or form heterodimers and trimers acting in one or more repair pathways. One hypothesis si that the human Rad51- related proteins, like yeast Rad55 and Rad57, function by physically cooperating with Rad51 in DNA repair. This hypothesis will be tested by determining how these proteins interact, and this information should give us important clues to their functions. Our goals are to more rigorously examine these interactions and to determine their biological significance. Co-immunoprecipitation (co-IP) and cytological localization experiments are proposed to extend our yeast two-hybrid results. HsRad51 focus induced by DNA damage will be characterized to see if they contain other Rad51-related proteins. Gel filtration studies will determine if these is one large complex or distinct hetero- multimers . Modified yeast two-hybrid experiment swill determine which pairs of protein interactions can occur simultaneously, and which occur competitively or cooperatively. A major strength of two-hybrid results is that is they rapidly suggest models that will then be tested by co-IP, gel filtration and immunolocalization experiments. The combined data will tell us whether or not these proteins form a recombinosome and if so, will elucidate its architecture. Two-hybrid deletion analysis will be used to map binding domains on XRCC2, XRCC2 and Rad51C, and interaction- defective mutations will be isolated. These interaction-defective mutations will be tested to see if they can complement knockout mutations and/or if they may act as dominant negative alleles. An interaction between Rad51C and IKK-beta has been observed in the two-hybrid system. IKK-beta is a kinase involved in the activation of NF-kappaB, and is part of a system that responds to x-rays and other stimuli. We will attempt to biochemically confirm the observed Rab51C-IKK-beta interaction, and also determine if Rad51C is phosphorylated. If so, we will test if phosphorylation changes in response to x-ray or MMC treatment, and examine its biological significance by analyzing mutant Rad51C that may not be phosphorylated on key residues.