Recombination Mechanisms in Yeast Cell Differentiation

描述（由申请人提供）：染色体双链断裂（DSBs）的修复对人类细胞的生存能力和基因组不稳定性的异常修复至关重要。这一建议继续研究DSB修复最常见的途径-基因转换-在模型系统中，出芽酵母酿酒酵母。通过使用半乳糖诱导的HO内切酶在种群的所有细胞中快速诱导单个DSB，可以对DSB修复进行详细分析。交配型（MAT）基因转换和异位重组都将被研究。对从重组细胞中分离的DNA进行物理分析，通过southern blot和PCR分析，可以鉴定重组的中间产物，染色质免疫沉淀（ChIP）允许人们在野生型和缺乏各种重组和DNA复制蛋白的细胞中流动重组蛋白的募集。我们建议继续分析同源重组的早期步骤，包括链入侵中间体的表征和突变重组蛋白的缺陷。将对基因转化过程中新DNA的合成进行详细分析，包括通过对HO诱导的重组温度敏感突变的物理监测来研究必要的复制蛋白。关键细胞分裂激酶Cdk1在重组的后续步骤中的作用将被研究。brdu标记DNA的分子梳理将用于分析重组过程中新DNA合成的程度和位置。对修复DNA复制的保真度进行分析。对活细胞中gfp标记的染色体位点的快速光镜分析将用于分析rad51介导的同源性搜索的动态；此外，不同供体序列之间的竞争实验将用于了解染色体内和染色体间重组中同源性搜索和链入侵的动态。一项重要的新工作将是研究一种新的重组执行检查点，其中两个DSB的突触以正确的方向结束，并且在相同的模板上，这似乎是激活完成基因转换所需的新DNA合成所必需的。本提案的第二个主要主题是了解重组增强子（RE）在MAT转换过程中调节两个备选供体之一的交配类型依赖选择中的作用。将对RE序列及其蛋白结合伙伴进行进一步的表征。将确定约束III号染色体左臂的顺式作用序列，包括HML。快速光学显微镜还将用于分析RE调控的染色体运动约束。

DESCRIPTION (provided by applicant): Repair of chromosome double-strand breaks (DSBs) is essential for viability in human cells and aberrant repair to genomic instability. This proposal continues the study of DSB repair by the most common pathway - gene conversion - in the model system, the budding yeast Saccharomyces cerevisiae. A detailed analysis of DSB repair is made possible by rapidly inducing a single DSB in all cells of the population, using a galactose-inducible HO endonuclease. Both mating-type (MAT) gene switching and ectopic recombination will be studied. Physical analysis of DNA isolated from cells undergoing recombination, by southern blot and PCR analysis, makes it possible to identify intermediates of recombination, and chromatin immunoprecipitation (ChIP) permits one to flow the recruitment of recombination proteins in both wild type and cells lacking various recombination and DNA replication proteins. It is proposed to continue our analysis of early steps in homologous recombination, including the characterization of strand invasion intermediates and the defects of mutant recombination proteins. A detailed analysis of new DNA synthesis during gene conversion will be performed, including a study of essential replication proteins by physical monitoring of HO- induced recombination temperature-sensitive mutations. The role of the key cell division kinase, Cdk1, in later steps of recombination will be studied. Molecular combing of BrdU-labeled DNA will be used to analyze the extent and location of new DNA synthesis during recombination. The fidelity of repair DNA replication will be analyzed. Very rapid light microscopic analysis of GFP-tagged chromosome sites in living cells will be used to analyze the dynamics of the Rad51-mediated search for homology; in addition competition experiments between different donor sequences will be used to understand the dynamics of homology searching and strand invasion in both intra- and interchromosomal recombination. A major new effort will be to investigate a novel recombination execution checkpoint, in which the synapsis of the two DSB ends in the correct orientation, and on the same template, appears to be required for activation of new DNA synthesis needed to complete gene conversion. A second major theme of this proposal is to understand the role of the Recombination Enhancer (RE) in regulating the mating-type dependent choice of one of two alternative donors during MAT switching. Further characterization of the RE sequence and its protein binding partners will be carried out. Cis-acting sequences that constrain the left arm of chromosome III, including HML, will be identified. Rapid light microscopy will also be used to analyze the constraints on chromosome movement regulated by RE.