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: 6867349
• 负责人: KENDALL L. KNIGHT
• 金额: $ 26.57万
• 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/6867349
• 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基因重组的分子机制。同源重组在维持基因组稳定性方面发挥着不可或缺的作用，是修复染色体双链断裂的重要机制。本研究重点关注人类 Rad52 蛋白 (HsRad52) 的功能组织以及 HsRad52 与其他人类重组蛋白之间的功能关系。 HsRad52 通过分子术语尚未定义的机制介导 HsRad51 的催化重组活性。 HsRad52 结合单链和双链 DNA，与 HsRad52 和 HsRPA 特异性相互作用，并形成环形寡聚物以及这些环的高阶组装体。我们最近在 HsRad52 内发现了一个专门调节这些高级结构形成的结构域，并证明这些结构与蛋白质功能相关。在这些研究中，我们将定义 HsRad52 中对其每种生化活性都很重要的残基，并将开展旨在了解每种活性在介导 HsRad51 催化链交换中的作用的重要性的研究。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，我们启动了使用人类细胞系的 RNA 干扰研究。 RNAi介导的内源基因产物的敲除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，这些方面是体内同源重组途径最佳功能所必需的。了解人类同源重组的分子机制原理对于创造具有所需特性的新型蛋白质具有深远的影响，这些蛋白质可用于预防由基因组不稳定引起的疾病和有益的基因操作。