Defining role of Fanconi anemia complementation group A protein in DNA repair

链间交联(ICL)是一种有害的DNA损伤，其中两条链是共价结合的 共同产生停滞的复制状态，对细胞产生细胞毒性。ICL的缺陷修复与 范可尼贫血综合征(FA)与癌症易感性。至少有17个FA基因已经被 标识为最新的。这项提案侧重于一种特定的FA蛋白，FANCA，FA核心的成员 在中突变的复合体在整个FA患者群体中的比例为%，并且没有明确的 功能。我们的初步研究表明，FANCA破坏(解开)DNA螺旋并识别DNA 复制叉中的链间交联损伤(ICL)。有趣的是，FANCA退火单链DNA和 也催化了链交换。此外，FANCA与RAD51、主要的 双链断裂修复中的重组酶。基于这些数据，我们假设FANCA直接 通过DNA破坏(解离)活动参与链间交联链的修复，并促进 随后通过催化链退火和交换来修复双链DNA断裂。为了 描述FANCA在DNA修复中的作用，我们将使用生物化学定义的体外系统，一种基于细胞的 DSB修复研究系统和活细胞成像系统实现了四个目的：1)勾画分子 FANCA如何催化DNA‘解旋’、单链退火和链交换的机制。2) 确定FANCA在识别和切断DNA链间交联物中的作用。3)学习生物学 FANCA在双链断裂修复中的作用4)研究DNA‘解开’的生理作用，单一- FANCA链退热和链交换活性在范可尼贫血病因中的作用。理解 FANCA在DNA修复中的作用不仅有助于全面阐明ICL修复过程，而且 也为范科尼贫血及其相关癌症的病因学提供了新的见解。

Interstrand crosslinks (ICLs) are deleterious DNA damages in which both strands are covalently bound together, generating a stalled replication state cytotoxic to cells. Defective repair of ICLs is associated with Fanconi Anemia syndrome (FA) and predisposition to cancer. At least seventeen FA genes have been identified up to date. This proposal focuses on a specific FA protein, FANCA, a member of the FA core complex that is mutated in 64% of the entire FA patient population and does not have a clear function. Our preliminary study shows that FANCA destabilizes (unwinds) DNA helix and recognizes DNA interstrand crosslink damage (ICL) in a replication fork. Intriguingly, FANCA anneals single-strand DNA and catalyzes strand exchange as well. Furthermore, FANCA synergistically interacts with Rad51, the major recombinase in double strand break (DSB) repair. Based on these data, we hypothesize that FANCA directly participates in repair of interstrand crosslinks through its DNA-destabilizing (`unwinding') activity and facilitates subsequent repair of double strand DNA breaks by catalyzing strand annealing and exchange. In order to delineate the role of FANCA in DNA repair, we will use a biochemically defined in vitro system, a cell-based DSB repair study system, and a living cell imaging system to accomplish four aims: 1) Delineate the molecular mechanism of how FANCA catalyzes DNA `unwinding', single-strand annealing, and strand exchange. 2) Determine the role of FANCA in recognition and incision of DNA interstrand crosslinks. 3) Study the biological role of FANCA in double strand break repair. 4) Examine the physiological role of the DNA `unwinding', single- strand annealing, and strand exchange activities of FANCA in the etiology of Fanconi anemia. Understanding the role of FANCA in DNA repair will not only contribute to the overall clarification of the ICL repair process, but also provide novel insights into the etiology of Fanconi anemia and its associated cancer.