ANALYSIS OF HUMAN RAD51 RELATED PROTEINS

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RAD51基因编码参与重组和DNA修复的链转移蛋白。在……里面。在酿酒酵母中，RAD51家族的相关蛋白还包括Rad55和Rad57，它们形成了与RAD51相互作用的异源二聚体。在人类细胞中，该家族中的六种蛋白(HsRad51、XRCC2、XRCC3、RAD51B/hRec2、Rad51C和RAD51D)可能参与重组修复和维持基因组稳定。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，要么形成了作用于一个或多个修复途径的异源二聚体和三聚体。一种假设是，人类RAD51相关蛋白，如酵母Rad55和Rad57，通过与RAD51在DNA修复中进行物理合作来发挥作用。这一假说将通过确定这些蛋白质如何相互作用来检验，这一信息应该会给我们提供关于它们功能的重要线索。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。提出了免疫共沉淀(co-IP)和细胞学定位实验来扩展我们的酵母双杂交结果。DNA损伤引起的HsRad51焦点将被鉴定，看看它们是否含有其他RAD51相关蛋白。凝胶过滤研究将确定这是一种大的、复杂的还是不同的杂多聚体。改良的酵母双杂交实验将确定哪些蛋白质相互作用可以同时发生，哪些是竞争发生的，哪些是合作发生的。双杂交结果的一个主要优点是，它们可以快速提出模型，然后通过联合IP、凝胶过滤和免疫定位实验进行测试。结合的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定位XRCC2、XRCC2和Rad51C上的结合结构域，并分离出相互作用缺陷突变。将对这些相互作用缺陷突变进行测试，以确定它们是否可以补充基因敲除突变和/或它们是否可能作为显性负等位基因。在双杂交体系中观察到了Rad51C和IKK-β之间的相互作用。IKK-β是一种参与激活核因子-kappaB的激酶，是对X射线和其他刺激做出反应的系统的一部分。我们将尝试用生化方法证实观察到的Rab51C-ikk-beta相互作用，并确定Rad51C是否被磷酸化。如果是这样的话，我们将测试磷酸化是否会随着X射线或MMC处理的反应而改变，并通过分析关键残基上可能不被磷酸化的突变体Rad51C来检测其生物学意义。

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.