(as stated in parent grant) The long-term goal of the research proposed here is to determine the molecular mechanism of homologous genetic recombination and DNA break repair. This objective is approached by a combination of genetic analysis of mutants and biochemical analysis of proteins and DNA from cells. This research uses the fission yeast Schizosaccharomyces pombe as well as the bacterium Escherichia coli and its phage lambda. All are widely studied, highly tractable model organisms with features common to all organisms, including humans. The studies are focused on meiotic recombination in S. pombe, whose high rates of recombination facilitate both genetic and biochemical analyses, and on the major (RecBCD) pathway of recombination and DNA break repair in bacteria. Building on past achievements, the research is currently focused on the following goals. 1) Determine how meiotic recombination is repressed in and near centromeres, to prevent chromosome missegregation, infertility, and birth defects. 2) Determine how the correct template (sister or homolog) is chosen for meiotic DNA break repair, to elucidate how crossing-over is controlled across the genome to favor proper meiotic chromosome segregation. 3) Determine how appropriate joint DNA molecules are formed, and inappropriate ones are avoided, to ensure high viability of meiotic products (gametes). 4) Determine the mechanism by which RecBCD enzyme interacts with its controlling site on DNA (the Chi hotspot), to allow high-definition analysis of bacterial recombination. 5) Determine how recently discovered small-molecule inhibitors block one or another RecBCD enzymatic activity, to be developed into potentially useful, sorely needed antibiotics. These goals will be attacked by a combination of genetic analysis of mutants, fluorescence microscopy of intracellular proteins and chromosomal sites, physical analysis of DNA intermediates from meiotic cells, and enzymatic and biophysical analyses of isolated proteins. The results of these studies will elucidate the molecular mechanism of recombination and DNA break repair as well as the controls on recombination that ensure that it occurs at the proper time and place along chromosomes. Recombination is important for faithful meiotic chromosome segregation, for error-free repair of frequently arising DNA double-strand breaks, and for generating cellular and organismal diversity. Aberrancies of recombination can generate chromosomal rearrangements, such as translocations, duplications, and deletions, which are often associated with or the cause of infertility, birth defects, and cancers. Inhibitors of bacterial DNA break repair are promising novel antibiotics, which are desperately needed to combat the ever- increasing prevalence of drug-resistant bacteria.
Program Narrative (Relevance) (as stated in parent grant) The DNA of our cells is frequently broken and must be faithfully repaired for life to continue. Failure of faithful DNA break-repair by genetic recombination leads to birth defects, infertility, and cancer. Inhibitors of DNA break-repair in bacteria can be developed into novel, sorely needed antibiotics to combat the ever- threatening emergence of drug-resistant bacteria.
