Recombination-mediated DNA repair in yeast

描述（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。这个过程中的缺陷可能会导致致命的后果，或者导致肿瘤细胞中典型的染色体重排。在这里，我们将通过应用生化、分子和遗传方法的组合在酵母中探索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介导一种特定于被捕获的复制分叉的修复形式。第一个目标应用一些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，测定核酸酶活性，并对共纯化蛋白进行鉴定和表征。通过DNA损伤诱导蛋白质和磷酸化的作用也将被检查。核酸酶识别底物的范围将通过设计和测定不同大小的底物来确定。在第二个目标中，我们将确定MMS4-MUS81在各种重组试验中的作用。与同源内切酶Radl-10的功能重叠将通过上位分析和过表达抑制来研究。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。第三个目标采用几种基因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负责绕过SGS1 MUS81要求的途径。我们还将寻找mus81的DNA损伤敏感性的抑制因子。最后，我们将使用一种新的合成致死筛选方法来鉴定需要MMS4-MUS81进行双链断裂修复的突变体。预计这些实验结果将揭示真核生物复制叉阻滞的性质和重组介导的DNA修复机制。

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.