THE ROLE OF BRCA2 & FANCD2 IN CHROMOSOME DAMAGE REPAIR

遗传研究表明，肿瘤抑制因子BRCA 2和FANCD 2蛋白是肿瘤发生的关键成员。 范可尼贫血途径DNA损伤反应，在染色体损伤修复中通过同源 重组BRCA 2结合DNA并与RAD 51重组酶和FANCD 2蛋白结合。 我们假设这些BRCA 2-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， 以依赖于RAD 51和DNA的方式，由BRCA 2衍生的多肽的活性 与BRCA 2结合。我们的研究项目将继续破译机械基础和后果 BRCA 2-配体相互作用的背景下，DNA断裂和交联修复反应。 为了实现我们的目标，破译BRCA 2和FANCD 2蛋白在染色体中的作用， 损伤修复，分子研究下的三个具体目标将进行。具体目标1： 通过检查DNA结合，对BRCA 2 DNA结合结构域进行详细的分子表征 特异性，检查三个OB折叠在DNA底物接合中的作用，确定其功能， OB 2附加的Tower结构域在DNA结合特异性中的作用，并评估OB折叠的影响 和塔突变的生物化学和基因。具体目标2侧重于BRCA 2的作用 RAD 51介导的同源重组中的羧基末端。在这里，我们将构建，纯化， 表征BRCA 2的功能性多肽，所述多肽包含其C-末端RAD 51结合结构域， 利用我们独特的生物化学系统来测试这个C末端结构域帮助牧羊人的假设， RAD 51的复合基板。具体目标3旨在测试有关调制的假设 BRCA 2 DNA结合和重组介体的功能由FANCD 2。为了实现这一目标，我们 将组装BRCA 2衍生多肽和FANCD 2的复合物，并将检查这些复合物 用于与Rad 51的DNA结合和功能相互作用。BRCA 2-FANCD 2复合物的意义 将通过构建和表征FANCD 2缺陷的BRCA 2突变体来确定 互动将与项目1、2和3以及核心项目合作开展基于细胞的功能测定。 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.