MAMMALIAN DOUBLE-STRAND BREAK AND RECOMBINATIONAL REPAIR

这项拟议的研究的目标是确定几种哺乳动物的结构和酶的作用 DNA双链断裂的同源重组修复(HR)中的蛋白质，并确定 这些蛋白质是如何调节自发性心率的。双链断裂是由化学物质和辐射引起的，它们会 在DNA复制过程中自发产生。双链断裂由HR和非同源末端连接修复 (NHEJ)。在HR中直接作用的蛋白质包括RAD51，5个RAD51类似物(XRCC2，XRCC3，RAD51B， RAD51C和RAD51D)、BRCA1和BRCA2。RAD51催化DNA链转移，因此 是人力资源流程的核心。然而，RAD51很难在哺乳动物细胞中进行研究，因为零突变是 对细胞和胚胎都是致命的。RAD51对偶线形成几个复合体，这些复合体在物理上 与RAD51和/或DNA相互作用，并被认为增强RAD51的活性。BRCA2与RAD51相互作用 和其他蛋白质，以及单链和双链DNA。因此，RAD51 Paralog和BRCA2 在管理人力资源效率和结果方面具有独特的地位。任何一项的缺陷和/或表达改变 这些蛋白质导致基因组不稳定和癌症易感性。我们最近证明了 XRCC3影响HR的早期和晚期，并且几个BRCA2功能域起主导作用 对DSB诱导的人细胞心率的负面影响。提出了两个具体的目标来调查(1) XRCC2、XRCC3和RAD51C在DSB诱导和自发HR中的结构和酶作用； (2)BRCA2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、RAD51和其他蛋白质相互作用的BRCA2结构域。这些 项目将帮助建立XRCC2、XRCC3、RAD51C、 和BRCA2在HR的早期和/或晚期，从而为HR的调节提供了新的见解， 维持基因组稳定，抑制肿瘤。因为DSB修复是细胞的关键决定因素 DNA损伤后的存活，从这些研究中获得的信息也将有助于 开发更有效的药物使肿瘤细胞对辐射和遗传毒性的DNA损伤敏感 化学制品。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。

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.