Defining the role of FANCD2 in homology-directed DNA Repair

描述（由申请方提供）：范可尼贫血（FA）是一种多基因常染色体隐性遗传综合征，其特征为多发性先天性异常、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损伤反应途径中起协同作用。该途径中的关键步骤是FANCD 2的单泛素化，导致其重新分布到核灶中。FA细胞对链间交联剂（如丝裂霉素C（MMC））非常敏感，但对电离辐射（IR）具有相对抗性，尽管这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FA通路响应于DSB而被普遍激活，但对于MMC诱导的损伤的损伤响应而言，FA通路是特别需要的。即使我们对FA通路的分子理解有了重大进展，但对其功能知之甚少。几种FA蛋白（A/C/E/F/G/L）是另一种FA蛋白FANCD 2响应DNA损伤时单泛素化所需的多亚基复合物的组分。在单泛素化后，激活的FANCD 2（FANCD 2-Ub）重新分布到核灶，在那里它与涉及同源依赖性修复（HDR）的几种蛋白质共定位。事实上，对于FANCD 2灶形成后FA通路的目的一无所知。我的假设是，由于FANCD 2本身没有明显的功能结构域，它通过影响已知的修复途径（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FANCD 2相互作用的其他修复途径，并确定FANCD 2如何影响MMC诱导的链间交联损伤修复中的这些途径。利用D 'Andrea实验室在细胞生物学方面的现有专业知识，以及我们全面的FA细胞库和与染色质生物学领域的领导者合作学习的应用技术，我提出了一种多方面的方法来分析FANCD 2蛋白在交联DNA修复中的作用。具体而言，我建议（i）表征在单泛素化和病灶形成后与FANCD 2结合的因子;（ii）鉴定在野生型FA途径活化后与FANCD 2差异结合的蛋白质，并选择FA患者细胞系;（iii）开发体外测定，以评估FANCD 2如何与DNA相互作用，以及它是否可以影响涉及BRCA 2介导的RAD 51丝形成和链交换的HDR修复测定。

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.