ANALYSIS OF HUMAN RAD51 RELATED PROTEINS

人RAD51相关蛋白的分析

基本信息

批准号：
6180346
负责人：
DAVID SCHILD
金额：
$ 35.38万
依托单位：
UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-03-01 至 2002-07-31
项目状态：
已结题

项目摘要

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.

越来越多的证据表明，重组修复在人类细胞中很重要，并且可能在预防癌症中起作用。酵母和人Rad51基因编码参与重组和DNA修复的链转移蛋白。在。 S. cerevisiae，相关蛋白的RAD51家族还包括RAD55和RAD57，它们形成与Rad51相互作用的异二聚体。在人类细胞中，该家族中的六种蛋白（HSRAD51，XRCC2，XRCC3，RAD51B/HREC2，RAD51C和RAD51D）可能参与重组修复并维持基因组稳定性。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，人蛋白要么是重组体的一部分，要么是在一个或多个修复途径中作用的异二聚体和三聚体。一种假设Si，即人Rad51相关蛋白（如酵母Rad55和Rad57）通过与DNA修复中的Rad51进行物理合作来起作用。该假设将通过确定这些蛋白质的相互作用来检验，并且该信息应为我们的功能提供重要的线索。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。提出了共免疫沉淀（CO-IP）和细胞学定位实验，以扩大我们的酵母两种杂交结果。 DNA损伤引起的HSRAD51焦点将被表征，以查看它们是否含有其他与Rad51相关的蛋白。凝胶过滤研究将确定这些是一个大型复合物还是不同的异源多组。改良的酵母双杂交实验swill确定可以同时发生哪些蛋白质相互作用，并且哪些蛋白质相互作用会发生竞争性或合作。两种杂交结果的主要强度是，它们迅速提出了模型，然后将通过Co-IP，凝胶过滤和免疫定位实验对其进行测试。组合数据将告诉我们这些蛋白质是否形成重组体，如果是的，则将阐明其架构。两个杂交缺失分析将用于绘制XRCC2，XRCC2和RAD51C上的结合结构域，并将分离相互作用的突变。这些相互作用缺陷的突变将进行测试，以查看它们是否可以补充敲除突变和/或它们是否可以充当主要的负等位基因。在两个杂交系统中已经观察到了Rad51c和Ikk-beta之间的相互作用。 Ikk-beta是一种参与NF-kappab激活的激酶，并且是对X射线和其他刺激做出反应的系统的一部分。我们将尝试在生化上确认观察到的RAB51C-IKK-β相互作用，并确定RAD51C是否磷酸化。如果是这样，我们将测试磷酸化是否会对X射线或MMC处理的响应发生变化，并通过分析可能不会在关键残基上磷酸化的突变体Rad51c来检查其生物学意义。