Recombination-mediated DNA repair in yeast

酵母中重组介导的 DNA 修复

• 批准号: 7060777
• 负责人: STEVEN J. BRILL
• 金额: $ 27.09万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7060777
• 关键词: DNA damage; DNA repair; DNA replication origin; Saccharomyces cerevisiae; biochemistry; cell cycle; chromatin; chromatography; endonuclease; enzyme activity; enzyme structure; enzyme substrate complex; fungal genetics; fungal proteins; gene targeting; genetic recombination; genetic screening; immunoprecipitation; matrix assisted laser desorption ionization; mutant; phosphorylation; protein purification; recombinant proteins; transcription factor

DESCRIPTION (provided by applicant): Homologous DNA recombination plays an essential role in repairing double strand breaks and stabilizing stalled replication forks. Defects in this process may have fatal consequences or result in chromosomal rearrangements typical of those found in tumor cells. Here we will explore the role of MMS4-MUS81 in recombination-mediated DNA repair by applying a combination of biochemical, molecular, and genetic approaches in yeast. Mms4-Mus81 was identified in yeast as a structure-specific endonuclease that functionally overlapped with the helicase-topoisomerase complex, Sgs1Top3. Our working hypothesis is that Mms4-Mus81 mediates a form of repair specific to arrested replication forks. The first Aim applies a number of biochemical approaches to determine the nuclease's structure, regulation and substrate-specificity. The native complex will be purified from yeast extracts, assayed for nuclease activities, and co-purifying proteins will be identified and characterized. Induction of the protein by DNA damage and the role of phosphorylation will also be examined. The range of substrates recognized by the nuclease will be determined by designing and assaying substrates of varying size. In the second Aim we will determine the role of MMS4-MUS81 in a variety of recombination assays. Functional overlap with the homologous endonuclease Radl-10 will be investigated using epistasis analysis and suppression by overexpression. Chromatin immunoprecipitation will be used to to search for the presence of Mms4-Mus81 at sites of DNA repair and replication fork pausing in-vivo. The third Aim employs several genetic screens to identify new recombinational repair genes. We will identify the pathway responsible for bypassing the SGS1 MUS81 requirement by performing an SGS1 MUS81 RAD51 synthetic-lethal screen. We will also search for suppressors of mus81' s DNA damage sensitivity. Finally, we will use a novel variation of the synthetic-lethal screen to identify mutants that require MMS4-MUS81 for double strand break repair. It is expected that the results of these experiments will shed light on the nature of replication fork arrest and the mechanism of recombination-mediated DNA repair in eukaryotes.

描述（由申请人提供）：同源DNA重组在修复双链断裂和稳定停滞的复制叉中起着至关重要的作用。此过程中的缺陷可能会带来致命的后果或导致肿瘤细胞中典型的染色体重排。在这里，我们将通过在酵母中应用生化，分子和遗传方法的组合来探讨MMS4-MUS81在重组介导的DNA修复中的作用。 MMS4-MUS81在酵母中被鉴定为特定于结构的内核酸酶，该核酸酶在功能上与解旋酶 - 昆虫异构酶复合物SGS1TOP3重叠。我们的工作假设是MMS4-MUS81介导了一种特定于被捕的复制叉的修复形式。第一个目的采用了许多生化方法来确定核酸酶的结构，调节和底物特异性。天然复合物将从酵母提取物中纯化，分析用于核酸酶活性，并将鉴定和表征共纯化的蛋白质。还将检查通过DNA损伤诱导蛋白质和磷酸化的作用。核酸酶识别的底物的范围将通过设计和分析大小变化的底物来确定。在第二个目标中，我们将确定MMS4-MUS81在各种重组测定中的作用。将使用上科学分析和通过过表达来研究与同源性内切酶RADL-10的功能重叠。染色质免疫沉淀将用于在DNA修复和复制叉子的位置搜索MMS4-MUS81的存在。第三个目标采用多个遗传筛选来鉴定新的重组修复基因。我们将通过执行SGS1 MUS81 RAD51合成致命筛选来确定负责绕过SGS1 MUS81需求的途径。我们还将搜索MUS81 DNA损伤敏感性的抑制剂。最后，我们将使用合成致命筛选的新型变化来识别需要MMS4-MUS81进行双链断裂修复的突变体。预计这些实验的结果将阐明复制叉停滞的性质以及真核生物中重组介导的DNA修复的机制。