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

链间交联（ICLs）是DNA双链共价结合的有害损伤 一起产生对细胞有细胞毒性的停滞复制状态。ICL的修复缺陷与 范可尼贫血综合征（FA）和癌症易感性。至少有17个FA基因被 识别到最新。该提案重点关注一种特定的FA蛋白，FANCA，FA核心的成员 在整个FA患者群体的64%中发生突变并且没有明确的 功能我们的初步研究表明，FANCA使DNA螺旋不稳定（解旋）并识别DNA 复制叉中链间交联损伤（ICL）。有趣的是，FANCA退火单链DNA， 也催化链交换。此外，FANCA与Rad 51协同相互作用， 双链断裂（DSB）修复中的重组酶。基于这些数据，我们假设FANCA直接 通过其DNA去稳定（“解旋”）活性参与修复链间交联，并促进 随后通过催化链退火和交换修复双链DNA断裂。为了 为了描述FANCA在DNA修复中的作用，我们将使用一种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，以实现四个目的：1）描绘分子 FANCA如何催化DNA“解旋”、单链退火和链交换的机制。（二） 确定FANCA在识别和切割DNA链间交联中的作用。3)研究其生物学 FANCA在双链断裂修复中的作用。4)研究DNA“解旋”的生理作用，单个- 链退火和链交换活动的FANCA在范可尼贫血的病因。理解 FANCA在DNA修复中的作用不仅有助于ICL修复过程的整体澄清， 还提供了新的见解范可尼贫血及其相关癌症的病因。

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.