Role of Human Rad52 and Rad51- paralogs in DNA Repair

人类 Rad52 和 Rad51- 旁系同源物在 DNA 修复中的作用

• 批准号: 7059436
• 负责人: KENDALL L. KNIGHT
• 金额: $ 26.01万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7059436
• 关键词: DNA binding protein; DNA repair; RNA interference; biochemistry; cell line; enzyme linked immunosorbent assay; gene targeting; genetic recombination; genetic screening; immunofluorescence technique; molecular assembly /self assembly; mutant; protein protein interaction; protein structure function; recombinant proteins; transfection /expression vector

DESCRIPTION (provided by applicant): The goal of these studies is to understand the molecular mechanism of homologous genetic recombination in human cells. Homologous recombination plays an integral role in maintaining genomic stability and is an important mechanism for the repair of chromosomal double-strand breaks. This study focuses on the functional organization of the human Rad52 protein (HsRad52) as well as the functional relationships between HsRad52 and other human recombination proteins. HsRad52 mediates the catalytic recombination activity of HsRad51 via a mechanism that has yet to be defined in molecular terms. HsRad52 binds both single and double-stranded DNA, interacts specifically with HsRad52 and HsRPA, and forms both ringshaped oligomers as well as higher-order assemblies of these rings. We have recently identified a domain within HsRad52 that specifically regulates formation of these higher-order structures and have demonstrated that these structures are relevant to protein function. In these studies we will define residues within HsRad52 that are important for each of its biochemical activities, and will carry out studies designed to understand the importance of each of these activities with regard to their role in mediating HsRad51-catalyzed strand exchange. An additional aim of our work is to define the molecular nature of the functional relationships between HsRad52 and the HsRad51 paralog proteins. For this work we have initiated RNA interference studies using human cell lines. RNAi-mediated knock down of endogenous gene products allows us to assess the function of mutant versions of the corresponding transgene for which we have mechanistic information from biochemical studies of that mutant protein. This combination of methods will provide important insights into specific biochemical aspects of HsRad52 and HsRad51 paralog proteins that are required for optimal function of the homologous recombination pathway in vivo. Understanding the molecular mechanistic principles of homologous recombination in humans has far-reaching effects for creating novel proteins with desired properties that may be used for prevention of diseases resulting from genomic instability and for beneficial genetic manipulation.

描述（由申请人提供）：这些研究的目的是了解人类细胞中同源遗传重组的分子机制。同源重组在维持基因组稳定性方面起着不可或缺的作用，并且是修复染色体双链断裂的重要机制。这项研究的重点是人Rad52蛋白（HSRAD52）的功能组织以及HSRAD52和其他人重组蛋白之间的功能关系。 HSRAD52通过一种尚未用分子术语定义的机制介导HSRAD51的催化重组活性。 HSRAD52结合了单链DNA，与HSRAD52和HSRPA特别相互作用，并形成了这些环的环形低聚物以及高阶组件。我们最近确定了HSRAD52中的一个域，该域专门调节这些高阶结构的形成，并证明这些结构与蛋白质功能有关。在这些研究中，我们将定义HSRAD52中的残基对每种生物化学活动都很重要，并将进行旨在了解每种活动在介导HSRAD51催化的链链交换中的作用的重要性。我们工作的另一个目的是定义HSRAD52和HSRAD51旁系同源蛋白之间功能关系的分子性质。对于这项工作，我们使用人细胞系开始了RNA干扰研究。 RNAi介导的内源基因产物的敲低使我们能够评估相应转基因的突变版本的功能，我们从该突变蛋白的生化研究中获得了机械信息。这种方法的组合将为HSRAD52和HSRAD51旁系同源蛋白的特定生化方面提供重要的见解，这些蛋白是体内同源重组途径所需的最佳功能所必需的。了解人类同源重组的分子机理原理具有深远的影响，可以创建具有所需特性的新型蛋白质，这些蛋白质可用于预防由基因组不稳定性和有益的遗传操纵引起的疾病。