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: 7548149
• 负责人: STEVEN P MARGOSSIAN
• 金额: $ 13.15万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7548149
• 关键词: BRCA2 gene; Behavior; Binding; Biological Assay; Biology; Cell Line; Cells; Cellular biology; Chromatin; Collaborations; Complex; Congenital Abnormality; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA lesion; Disease; Doctor of Medicine; Doctor of Philosophy; FANCD2 protein; Fanconi anemia protein; Fanconi' s Anemia; Filament; Genes; Genetic Recombination; In Vitro; Ionizing radiation; Learning; Lesion; Malignant Neoplasms; Mediating; Mitomycin; Molecular; Mono-S; Monoubiquitination; Mutagens; Nuclear; Pancytopenia; Pathway interactions; Patients; Phenotype; Predisposition; Process; Protein Binding; Proteins; Purpose; Resistance; Role; Syndrome; TNFRSF5 gene; Techniques; Ubiquitination; crosslink; in vitro Assay; mutant; novel; repaired; repository; response

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（由申请人提供）：Fanconi贫血（FA）是一种多基因常染色体隐性综合征，其特征是多种先天性异常，进行性骨髓衰竭和癌症易感性。越来越多的证据表明，新型DNA损伤反应途径中的FA蛋白合作。这一途径的关键步骤是FANCD2的单泛素化，从而将其重新分配到核灶中。 FA细胞对链间交联敏感，例如丝裂霉素C（MMC），但对电离辐射（IR）的抗性相对抗性，尽管两种诱变者都会产生双链断裂（DSB）并激活FA途径。这表明，尽管它响应DSB，但它的一般激活。 FA途径是针对MMC诱导病变的损伤反应所必需的。即使在我们对FA途径的分子理解方面取得了重大进展，对其功能的了解知之甚少。几种FA蛋白（A/C/E/F/G/L）是另一种FA蛋白单泛素化所需的多生成复合物的成分，即响应DNA损伤的FANCD2。单次泛素化后，活化的fancd2（fancd2-ub）重新分布为核灶，其中它与涉及同源依赖性修复（HDR）的几种蛋白质共定位。实际上，在FANCD2 FOCI形成后FA途径的目的几乎一无所知。我的假设是，没有明显的功能结构域，FANCD2通过影响已知的修复途径的功能（例如HDR途径）来促进DNA损伤的修复。为了解决FA途径的功能，必须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的身份，并确定FANCD2如何影响这些途径在MMC诱导的链间交叉链接病变中。利用D'Andrea实验室中现有的专业知识，以及我们的全面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习的技术，我提出了一种多方面的方法来分析FANCD2蛋白在交联DNA修复中的FANCD2蛋白的作用。具体而言，我建议（i）表征单泛素化和焦点形成后与fancd2结合的因素； （ii）鉴定在野生型和选择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与DNA相互作用，以及它是否会影响涉及BRCA2介导的RAD51细丝形成和链交换的HDR修复测定法。