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. ? ? ?
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