MAMMALIAN DOUBLE-STRAND BREAK AND RECOMBINATIONAL REPAIR

哺乳动物双链断裂和重组修复

• 批准号: 7123263
• 负责人: Jac A Nickoloff
• 金额: $ 2.04万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7123263
• 关键词: DNA binding protein; DNA damage; DNA repair; adenosinetriphosphatase; binding proteins; brca gene; cell cycle proteins; cell line; chromosomes; enzyme activity; gene expression; gene mutation; genetic markers; genetic recombination; genetic transcription; neoplasm /cancer genetics; nucleic acid sequence; polymerase chain reaction; protein protein interaction; protein structure function; restriction fragment length polymorphism; southern blotting; transcription factor

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.

描述（由申请人提供）：拟议研究的目的是确定几种哺乳动物蛋白在DNA双链断裂（DSB）同源重组修复（HR）中的结构和酶促作用，并确定这些蛋白如何调节自发HR。 DSB是由化学物质和辐射诱导的，并且在DNA复制过程中自发产生。 DSB通过人力资源和非同源最终连接（NHEJ）修复。在HR中具有直接作用的蛋白质包括RAD51，五个RAD51旁系同源物（XRCC2，XRCC3，RAD51B，RAD51C和RAD51D），BRCA1和BRCA1。 RAD51催化DNA链的转移，因此是HR过程的核心。然而，由于无效突变体是细胞和胚胎致死的，因此在哺乳动物细胞中很难研究RAD51。 RAD51旁系同源物形成几个复合物，这些复合物与Rad51和/或DNA物理相互作用，并被认为增强了Rad51活性。 BRCA2与RAD51和其他蛋白质以及单链DNA相互作用。因此，RAD51旁系同源物和BRCA2是独特的定位，以调节HR效率和结果。这些蛋白质中任何一种的缺陷和/或改变表达会导致基因组不稳定性和癌症易感性。我们最近证明XRCC3影响了HR的早期和晚期，并且几个BRCA2功能域对人类细胞中DSB诱导的HR具有显着的负面影响。提出了两个具体的目的，以研究（1）XRCC2，XRCC3和RAD51C在DSB诱导的和自发的HR中的结构和酶促作用； （2）BRCA2在DSB诱导和自发的HR中的几个功能域的作用，包括与单链DNA，RAD51和其他蛋白质相互作用的BRCA2域。这些项目将有助于在HR的早期和/或晚期XRCC2，XRCC3，RAD51C和BRCA2的特定结构或酶促结构域建立角色，从而为HR的调节，基因组稳定性的维持和抑制提供新的见解。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还将促进更有效的药物的发展，以使肿瘤细胞通过辐射和遗传毒性化学物质对DNA损伤敏感。 HR机制和人力资源调节蛋白的研究还将促进更有效的基因靶向和基因治疗系统的发展。