MAMMALIAN DOUBLE-STRAND BREAK AND RECOMBINATIONAL REPAIR

描述（由申请人提供）：拟议研究的目标是确定几种哺乳动物蛋白质在DNA双链断裂（DSB）的同源重组修复（HR）中的结构和酶作用，并确定这些蛋白质如何调节自发HR。DSB是由化学品和辐射诱导的，它们在DNA复制过程中自发产生。DSB通过HR和非同源末端连接（NHEJ）修复。在HR中起直接作用的蛋白质包括RAD 51、五种RAD 51旁系同源物（XRCC 2、XRCC 3、RAD 51 B、RAD 51 C和RAD 51 D）、BRCA 1和BRCA 2。RAD 51催化DNA链转移，因此对HR过程至关重要。然而，RAD 51很难在哺乳动物细胞中研究，因为无效突变体是细胞和胚胎致死的。RAD 51旁系同源物形成几种复合物，这些复合物与RAD 51和/或DNA物理相互作用，并被认为增强RAD 51活性。BRCA 2与RAD 51和其他蛋白质以及单链和双链DNA相互作用。因此，RAD 51旁系同源物和BRCA 2独特地定位于调节HR效率和结果。任何这些蛋白质的缺陷和/或改变的表达导致基因组不稳定性和癌症易感性。我们最近证明XRCC 3影响HR的早期和晚期阶段，并且几个BRCA 2功能结构域对人细胞中DSB诱导的HR具有显性负效应。提出了两个具体的目的来研究（1）XRCC 2，XRCC 3和RAD 51 C在DSB诱导和自发HR中的结构和酶促作用;（2）BRCA 2的几个功能结构域在DSB诱导和自发HR中的作用，包括与单链和双链DNA，RAD 51和其他蛋白质相互作用的BRCA 2结构域。这些项目将有助于确定XRCC 2、XRCC 3、RAD 51 C和BRCA 2的特定结构或酶结构域在HR早期和/或晚期阶段的作用，从而为HR的调节、基因组稳定性的维持和肿瘤抑制提供新的见解。由于DSB修复是DNA损伤后细胞存活的关键决定因素，因此从这些研究中获得的信息也将有助于开发更有效的药物，以使肿瘤细胞对辐射和遗传毒性化学品引起的DNA损伤敏感。 对HR机制和HR调节蛋白的研究也将促进更有效的基因靶向和基因治疗系统的发展。

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the structural and enzymatic roles of several mammalian proteins in homologous recombinational repair (HR) of DNA double-strand breaks (DSBs), and to determine how these proteins regulate spontaneous HR. DSBs are induced by chemicals and radiation and they arise spontaneously during DNA replication. DSBs are repaired by HR and by nonhomologous end-joining (NHEJ). Proteins with direct roles in HR include RAD51, five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C, and RAD51D), BRCA1, and BRCA2. RAD51 catalyzes DNA strand transfer and is therefore central to HR processes. However, RAD51 is difficult to study in mammalian cells because null mutants are cell- and embryo-lethal. The RAD51 paralogs form several complexes, and these complexes physically interact with RAD51 and/or DNA, and are thought to augment RAD51 activity. BRCA2 interacts with RAD51 and other proteins, as well as single- and double-stranded DNA. The RAD51 paralogs and BRCA2 are thus uniquely positioned to regulate HR efficiency and outcome. Defects and/ or altered expression of any of these proteins result in genome instability and cancer predisposition. We recently demonstrated that XRCC3 influences early and late stages of HR, and that several BRCA2 functional domains have dominant negative effects on DSB-induced HR in human cells. Two specific aims are proposed to investigate (1) the structural and enzymatic roles of XRCC2, XRCC3, and RAD51C in DSB-induced and spontaneous HR; and (2) the roles of several functional domains of BRCA2 in DSB-induced and spontaneous HR, including BRCA2 domains that interact with single- and double-stranded DNA, RAD51, and other proteins. These projects will help establish roles for specific structural or enzymatic domains of XRCC2, XRCC3, RAD51C, and BRCA2 in early and/or late stages of HR, and thereby provide new insight into the regulation of HR, maintenance of genome stability, and tumor suppression. Because DSB repair is a key determinant of cell survival following DNA damage, the information gained from these studies will also facilitate the development of more effective agents to sensitize tumor cells to DNA damage by radiation and genotoxic chemicals. Studies of HR mechanisms and HR regulatory proteins will also facilitate the development of more effective gene targeting and gene therapy systems.