Recombination-mediated DNA repair in yeast
酵母中重组介导的 DNA 修复
基本信息
- 批准号:6887689
- 负责人:
- 金额:$ 27.74万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2003
- 资助国家:美国
- 起止时间:2003-05-01 至 2007-04-30
- 项目状态:已结题
- 来源:
- 关键词:DNA damageDNA repairDNA replication originSaccharomyces cerevisiaebiochemistrycell cyclechromatinchromatographyendonucleaseenzyme activityenzyme structureenzyme substrate complexfungal geneticsfungal proteinsgene targetinggenetic recombinationgenetic screeningimmunoprecipitationmatrix assisted laser desorption ionizationmutantphosphorylationprotein purificationrecombinant proteinstranscription factor
项目摘要
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发挥着重要作用。这一过程中的缺陷可能会产生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索 MMS4-MUS81 在重组介导的 DNA 修复中的作用。 Mms4-Mus81 在酵母中被鉴定为一种结构特异性核酸内切酶,其功能与解旋酶-拓扑异构酶复合物 Sgs1Top3 重叠。我们的工作假设是 Mms4-Mus81 介导一种针对停滞复制叉的特定修复形式。第一个目标应用多种生化方法来确定核酸酶的结构、调节和底物特异性。将从酵母提取物中纯化天然复合物,测定核酸酶活性,并对共纯化蛋白质进行鉴定和表征。还将检查 DNA 损伤对蛋白质的诱导以及磷酸化的作用。核酸酶识别的底物范围将通过设计和分析不同大小的底物来确定。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。将使用上位性分析和过表达抑制来研究与同源核酸内切酶 Radl-10 的功能重叠。染色质免疫沉淀将用于在体内 DNA 修复和复制叉暂停位点寻找 Mms4-Mus81 的存在。第三个目标采用多种遗传筛选来识别新的重组修复基因。我们将通过执行 SGS1 MUS81 RAD51 合成致死筛选来确定绕过 SGS1 MUS81 要求的途径。我们还将寻找 mus81 DNA 损伤敏感性的抑制因子。最后,我们将使用合成致死筛选的新变体来识别需要 MMS4-MUS81 进行双链断裂修复的突变体。预计这些实验的结果将揭示真核生物复制叉停滞的本质和重组介导的 DNA 修复机制。
项目成果
期刊论文数量(0)
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STEVEN J. BRILL其他文献
STEVEN J. BRILL的其他文献
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{{ truncateString('STEVEN J. BRILL', 18)}}的其他基金
Mechanism of the BLM/Sgs1 Helicase Complex
BLM/Sgs1 解旋酶复合物的机制
- 批准号:
8292698 - 财政年份:2012
- 资助金额:
$ 27.74万 - 项目类别:
Mechanism of the BLM/Sgs1 Helicase Complex
BLM/Sgs1 解旋酶复合物的机制
- 批准号:
8623139 - 财政年份:2012
- 资助金额:
$ 27.74万 - 项目类别:
Mechanism of the BLM/Sgs1 Helicase Complex
BLM/Sgs1 解旋酶复合物的机制
- 批准号:
8602662 - 财政年份:2012
- 资助金额:
$ 27.74万 - 项目类别:
Mechanism of the BLM/Sgs1 Helicase Complex
BLM/Sgs1 解旋酶复合物的机制
- 批准号:
8464168 - 财政年份:2012
- 资助金额:
$ 27.74万 - 项目类别:
Genomic Stability and RecQ DNA Helicases in Yeast
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- 批准号:
7035787 - 财政年份:1999
- 资助金额:
$ 27.74万 - 项目类别:
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