The long-term goal of the proposed research is to determine the molecular mechanism of homologous genetic recombination and DNA break repair. This goal is approached by studying hotspots of recombination, which stimulate a critical, rate-limiting step of recombination. In the bacterium Escherichia coli, studies will focus on Chi hotspots, which stimulate the major (RecBCD) pathway of recombination and DNA break repair. In the fission yeast Schizosaccharomyces pombe, studies will focus on mutationally created hotspots in the ade6 gene and naturally occurring hotspots across the entire genome. These microbes are especially amenable for genetic and biochemical analyses, but in many ways their recombination mimics that of humans.
The specific aims are 1) to elucidate the complex interaction of Chi hotspots and RecBCD enzyme, with special emphasis on testing specific hypotheses of RecBCD's conformational change at Chi which we have recently demonstrated, and 2) to elucidate the determinants of hotspots of meiotic DSB formation across the S. pombe genome, with special emphasis on the interaction of hotspots and their evolution.
These aims will be achieved by a combination of biochemistry and electron microscopy with purified components, and genetics, DNA analysis, and fluorescence microscopy with intact cells. The results of these studies will elucidate both the mechanism of recombination and its regulation along chromosomes and during the organism's life cycle. Recombination is important in the faithful repair of DSBs in chromosomes and in the faithful segregation of chromosomes during meiosis. Aberrancies of recombination and DNA break repair are responsible for chromosomal aberrations that are associated with and apparent causes of cancer, birth defects, and certain hereditary diseases. RecBCD and closely related enzymes are widely distributed among bacteria but not eukaryotes and may therefore be good targets for a new class of critically needed antibiotics. Thus, the basic research proposed here will add to the foundations for understanding, diagnosing, preventing, and curing human disease.
Repair of broken DNA and the genetic recombination that can result from this repair are important for the health of cells and the evolution of species. Aberrancies of repair and recombination can result in cancer or birth defects. The research proposed here will help understand the causes of these diseases and may lead to ways to cure or prevent them, including the possible discovery of new antibacterial drugs that interfere with proper DNA repair.
|Amundsen, Susan K; Sharp, Jake W; Smith, Gerald R (2016) RecBCD Enzyme ""Chi Recognition"" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context. Genetics 204:139-52|
|Taylor, Andrew F; Amundsen, Susan K; Smith, Gerald R (2016) Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots. Nucleic Acids Res 44:8216-28|
|Nambiar, Mridula; Smith, Gerald R (2016) Repression of harmful meiotic recombination in centromeric regions. Semin Cell Dev Biol 54:188-97|
|Hyppa, Randy W; Fowler, Kyle R; Cipak, Lubos et al. (2014) DNA intermediates of meiotic recombination in synchronous S. pombe at optimal temperature. Nucleic Acids Res 42:359-69|
|Taylor, Andrew F; Amundsen, Susan K; Guttman, Miklos et al. (2014) Control of RecBCD enzyme activity by DNA binding- and Chi hotspot-dependent conformational changes. J Mol Biol 426:3479-99|
|Zanders, Sarah E; Eickbush, Michael T; Yu, Jonathan S et al. (2014) Genome rearrangements and pervasive meiotic drive cause hybrid infertility in fission yeast. Elife 3:e02630|
|Fowler, Kyle R; Sasaki, Mariko; Milman, Neta et al. (2014) Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome. Genome Res 24:1650-64|
|MartÃn-Castellanos, Cristina; Fowler, Kyle R; Smith, Gerald R (2013) Making chromosomes hot for breakage. Cell Cycle 12:1327-8|
|Fowler, Kyle R; Gutierrez-Velasco, Susana; Martin-Castellanos, Cristina et al. (2013) Protein determinants of meiotic DNA break hot spots. Mol Cell 49:983-96|
|Amundsen, Susan K; Spicer, Timothy; Karabulut, Ahmet C et al. (2012) Small-molecule inhibitors of bacterial AddAB and RecBCD helicase-nuclease DNA repair enzymes. ACS Chem Biol 7:879-91|
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