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 to effect mating-type (MAT) gene switching. Physical analysis of DNA solated 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, ncluding the characterization of strand invasion intermediates and the defects of mutant recombination proteins. The dynamics of homology searching will be investigated, using microscopic analysis of fluorescently-tagged segments adjacent to chromosomal regions undergoing recombination, as well as by Chip. Special attention will be given to the role of the cis-acting Recombination Enhancer. A detailed analysis of new DNA synthesis during gene conversion will be performed, including a study of recruitment of DNA polymerases to the strand invasion intermediate. Another finding that will be pursued concerns a 1000-fold increase in the rate of mutagenesis by template switching during gene conversion. The role of DNA processivity factors and helicases will be examined to identify the basis of template switching. In addition, the analysis of changes in chromatin structure accompanying MST switching will be pursued, using a modified MAT locus that allows the analysis of chromatin alterations and reestablishment during repair and the analysis of chromatin chaperones and remodelers. 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. The role of the phospho-threonine binding HA domain of Fkhl, which has been shown to be essential for donor preference when bound at RE, will be investigated. Identification of the phospho-threonine target in the region around the DSB will be carried out, both by characterization of the FHA domain and by analysis of DSB-dependent proteins that associate with the FHA domain.
NIH ExPorter
NIH ExPorter