Recombination Mechanisms in Yeast Cell Differentiation

描述（由申请方提供）：染色体双链断裂（DSB）的修复对于人细胞的活力和基因组不稳定性的异常修复至关重要。该建议继续研究DSB修复的最常见的途径-基因转换-在模型系统中，芽殖酵母酿酒酵母。通过使用半乳糖诱导型HO内切核酸酶在群体的所有细胞中快速诱导单个DSB，使得DSB修复的详细分析成为可能。交配型（MAT）基因转换和异位重组将进行研究。通过Southern印迹和PCR分析，对从经历重组的细胞中分离的DNA进行物理分析，使得有可能鉴定重组的中间体，并且染色质免疫沉淀（ChIP）允许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。建议继续我们的同源重组的早期步骤的分析，包括链侵入中间体和突变重组蛋白的缺陷的表征。将进行基因转换过程中新DNA合成的详细分析，包括通过物理监测HO诱导的重组温度敏感性突变来研究基本复制蛋白。将研究关键细胞分裂激酶Cdk 1在重组后期的作用。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。将分析修复DNA复制的保真度。非常快速的光显微镜分析的GFP标记的染色体位点在活细胞中将被用来分析的Rad 51介导的搜索同源性的动力学;此外，不同的供体序列之间的竞争实验将被用来了解的同源性搜索和链入侵的动力学内和染色体间重组。一个主要的新努力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，并且在同一模板上，似乎是激活完成基因转换所需的新DNA合成所必需的。该提案的第二个主要主题是了解在MAT转换期间，增强子（RE）在调节两个备选供体之一的交配型依赖性选择中的作用。将对RE序列及其蛋白结合伴侣进行进一步表征。将鉴定限制染色体III左臂（包括HML）的顺式作用序列。快速光学显微镜也将用于分析RE调控的染色体运动的限制。

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.