Defining the role of FANCD2 in homology-directed DNA Repair

描述(申请人提供)：Fanconi贫血(FA)是一种多基因常染色体隐性遗传综合征，以多种先天性异常、进行性骨髓衰竭和癌症易感性为特征。越来越多的证据表明，FA蛋白在一种新的DNA损伤反应途径中相互作用。FANCD2途径中的一个关键步骤是FANCD2的单泛素化，导致其重新分布到核内。FA细胞对丝裂霉素C(MMC)等链间交联剂非常敏感，但对电离辐射(IR)相对耐受，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通路对DSB的反应是普遍激活的，但FA通路对于MMC诱导的损伤反应是特殊需要的。尽管我们对FA途径的分子理解有了很大的进展，但人们对其功能知之甚少。一些FA蛋白(A/C/E/F/G/L)是另一种FA蛋白FANCD2对DNA损伤的单素化所需的多亚单位复合体的组成部分。在单素化后，激活的FANCD2(FANCD2-Ub)重新分布到核焦点，在那里它与几个参与同源依赖修复(HDR)的蛋白质共定位。FANCD2病灶形成后FA通路的作用几乎一无所知。我的假设是，FANCD2本身没有明显的功能结构域，通过影响已知的修复途径的功能，如HDR途径，促进DNA损伤的修复。为了解决FA途径的功能，必须识别其他基因产物的身份，并潜在地识别可能与FANCD2相互作用的其他修复途径，并确定FANCD2在MMC诱导的链间连锁损伤修复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，以及我们全面的FA细胞库，并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修复中的作用。具体地说，我建议(I)鉴定在单素化和焦点形成后与FANCD2结合的因素；(Ii)在野生型FA途径激活后识别与FANCD2有差异结合的蛋白质，并选择FA患者细胞系；(Iii)建立体外分析方法，评估FANCD2如何与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修复分析。

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.