THE ROLE OF BRCA2 & FANCD2 IN CHROMOSOME DAMAGE REPAIR

遗传学研究已发现肿瘤抑制基因BRCA2和FANCD2蛋白，FANCD2蛋白是 染色体损伤修复中DNA损伤反应的Fanconi贫血途径 重组。BRCA2与DNA结合，并与RAD51重组酶和FANCD2蛋白结合。 我们假设这些BRCA2-配体相互作用与染色体损伤修复密切相关。 与这一假设一致，我们的初步研究发现RAD51重组酶增强 BRCA2衍生多肽的活性，其活性依赖于RAD51和DNA 由BRCA2结合。我们的研究项目将继续破译其机理基础和后果。 在DNA断裂和交联修复反应的背景下BRCA2-配体相互作用的研究。 为了实现我们破译BRCA2和FANCD2蛋白在染色体中的作用的目标 损伤修复，将在三个特定目标下进行分子研究。在具体目标1中，我们将 通过检查DNA结合来对BRCA2 DNA结合域进行详细的分子表征 特异性，检测三个OB折叠在DNA底物结合中的作用，确定功能 OB2-附加塔结构域在DNA结合特异性中的作用，并评估OB折叠的影响 和Tower在生化和遗传上的突变。具体目标2侧重于BRCA2的作用 RAD51介导的同源重组中的羧基末端。在这里，我们将构建、净化和 鉴定BRCA2的C-末端RAD51结合域的功能多肽和 利用我们独特的生化系统来测试这个C-末端结构域帮助牧羊人 RAD51连接到重组底物上。特定目标3旨在测试有关调制的假设 FANCD2对BRCA2 DNA结合和重组的调节作用。为了达到这一目标，我们 将组装BRCA2衍生多肽和FANCD2的复合体，并将研究这些复合体 用于DNA结合和与RAD51的功能相互作用。BRCA2-FANCD2复合体的意义 将通过构建和鉴定在FANCD2中有缺陷的BRCA2突变体来确定 互动。将与项目1、2和3以及核心合作进行基于细胞的功能分析 C。

Genetic studies have implicated the tumor suppressor BRCA2 and the FANCD2 protein, a key member of the Fanconi anemia pathway of DNA damage response, in chromosome damage repair by homologous recombination. BRCA2 binds DNA and associates with the RAD51 recombinase and the FANCD2 protein. We hypothesize that these BRCA2-ligand interactions are germane for chromosome damage repair. Consistent with this hypothesis, our preliminary studies have found enhancement of the RAD51 recombinase activity by a polypeptide derived from BRCA2, in a manner that is dependent on both RAD51 and DNA binding by BRCA2. Our research project will continue to decipher the mechanistic bases and consequences of BRCA2-ligand interactions in the context of DNA break and crosslink repair reactions. To accomplish our goal of deciphering the role of the BRCA2 and FANCD2 proteins in chromosome damage repair, molecular studies under three specific aims will be carried out. In Specific Aim 1, we will conduct a detailed molecular characterization of the BRCA2 DNA binding domain by examining DNA binding specificity, examining the role of the three OB folds in DNA substrate engagement, determining the function of the OB2-appended Tower domain in DNA binding specificity, and also assessing the effects of OB fold and Tower mutations biochemically and genetically. Specific Aim 2 focuses on the role of the BRCA2 carboxyl-terminus in RAD51-mediated homologous recombination. Herein, we will construct, purify, and characterize functional polypeptides of BRCA2 that encompass its C-terminal RAD51 binding domain and employ our unique biochemical systems to test the hypothesis that this C-terminal domain helps shepherd RAD51 to the recombination substrate. Specific Aim 3 is designed to test hypotheses concerning modulation of the BRCA2 DNA binding and recombination mediator functions by FANCD2. To achieve this objective, we will assemble complexes of BRCA2-derived polypeptides and FANCD2 and will examine these complexes for DNA binding and functional interactions with Rad51. The significance of the BRCA2-FANCD2 complex will be ascertained by constructing and characterizing BRCA2 mutants that are defective in FANCD2 interaction. Cell-based functonal assays wil be carried out in collaboration with Projects 1, 2 and 3 and Core C.