Defining the role of FANCD2 in homology-directed DNA Repair

定义 FANCD2 在同源定向 DNA 修复中的作用

• 批准号: 6958977
• 负责人: STEVEN P MARGOSSIAN
• 金额: $ 13.15万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-05 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6958977
• 关键词: DNA damage; DNA repair; SDS polyacrylamide gel electrophoresis; congenital aplastic anemia; crosslink; immunoprecipitation; ionizing radiation; protein binding; protein sequence; protein structure function; ubiquitin

DESCRIPTION (provided by applicant): Fanconi Anemia (FA) is a multigenic autosomal recessive syndrome characterized by multiple congenital abnormalities, progressive bone marrow failure, and cancer susceptibility. Increasing evidence indicates the FA proteins cooperate in a novel DNA damage response pathway. A key step in this pathway is the monoubiquitination of FANCD2, resulting in its redistribution into nuclear foci. FA cells are exquisitely sensitive to interstrand cross-linkers, such as Mitomycin C (MMC), but relatively resistant to ionizing radiation (IR), although both mutagens generate double-strand breaks (DSBs) and activate the FA pathway. This suggests that, despite its general activation in response to DSBs; the FA pathway is specifically required for the damage response to MMC induced lesions. Even with major advances in our molecular understanding of the FA pathway, little is known about its function. Several FA proteins (A/C/E/F/G/L) are components of a multi-subunit complex required for the monoubiquitination of another FA protein, FANCD2 in response to DNA damage. Upon monoubiquitination, the activated FANCD2 (FANCD2-Ub) redistributes to nuclear foci where it colocalizes with several proteins implicated in homology-dependent repair (HDR). Virtually nothing is known about the purpose of the FA pathway following formation of FANCD2 foci. It is my hypothesis that, having no obvious functional domains of its own, FANCD2 facilitates repair of DNA damage by influencing the function of known repair pathways, such as the HDR pathway. To resolve the function of the FA pathway, it is essential to discern the identities of other gene products and, potentially, other repair pathways that may interact with FANCD2 and determine how FANCD2 influences these pathways in the repair of MMC induced interstrand cross-linked lesions. Taking advantage of the existing expertise in the D'Andrea lab in cell biology, and our comprehensive FA cell repository and applying techniques learned in collaboration with leaders in the field of chromatin biology, I propose a multifaceted approach to analyze the role of the FANCD2 protein in the repair of cross-linked DNA. Specifically, I propose to (i) Characterize the factors that bind to FANCD2 following monoubiquitination and foci formation; (ii) Identify proteins that bind differentially to FANCD2 following activation of the FA pathway in wild-type and select FA patient cell lines; (iii) Develop in vitro assays, to evaluate how FANCD2 interacts with DNA, and whether it can influence HDR repair assays involving BRCA2-mediated RAD51 filament formation and strand exchange.

描述（申请人提供）：范可尼贫血（FA）是一种多基因常染色体隐性遗传综合征，其特征为多种先天性异常、进行性骨髓衰竭和癌症易感性。越来越多的证据表明 FA 蛋白在一种新的 DNA 损伤反应途径中合作。该途径的关键步骤是 FANCD2 的单泛素化，导致其重新分布到核灶中。 FA 细胞对丝裂霉素 C (MMC) 等链间交联剂极其敏感，但对电离辐射 (IR) 相对抵抗，尽管两种诱变剂都会产生双链断裂 (DSB) 并激活 FA 途径。这表明，尽管它通常会响应 DSB 而激活； FA 途径是对 MMC 诱导病变的损伤反应所特别需要的。尽管我们对 FA 途径的分子理解取得了重大进展，但对其功能仍知之甚少。几种 FA 蛋白 (A/C/E/F/G/L) 是另一种 FA 蛋白 FANCD2 单泛素化所需的多亚基复合物的组成部分，以响应 DNA 损伤。单泛素化后，激活的 FANCD2 (FANCD2-Ub) 重新分布到核灶，与同源依赖性修复 (HDR) 相关的几种蛋白质共定位。事实上，对于 FANCD2 灶形成后 FA 途径的目的一无所知。我的假设是，FANCD2 本身没有明显的功能域，它通过影响已知修复途径（例如 HDR 途径）的功能来促进 DNA 损伤的修复。为了解决 FA 途径的功能，有必要辨别其他基因产物的身份，以及可能与 FANCD2 相互作用的其他修复途径，并确定 FANCD2 在修复 MMC 诱导的链间交联损伤中如何影响这些途径。利用 D'Andrea 实验室在细胞生物学方面的现有专业知识、我们全面的 FA 细胞存储库以及应用与染色质生物学领域的领导者合作学到的技术，我提出了一种多方面的方法来分析 FANCD2 蛋白的作用修复交联 DNA。具体来说，我建议 (i) 表征单泛素化和病灶形成后与 FANCD2 结合的因子； (ii) 鉴定在野生型和选择的 FA 患者细胞系中 FA 途径激活后与 FANCD2 差异结合的蛋白质； (iii) 开发体外测定，以评估 FANCD2 如何与 DNA 相互作用，以及它是否可以影响涉及 BRCA2 介导的 RAD51 丝形成和链交换的 HDR 修复测定。