Molecular Analysis of Hotspots of Genetic Recombination

Fred Hutchinson Cancer Research Center, Seattle WA

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Abstract

The long-term goal of the proposed research is to elucidate the molecular mechanism of homologous genetic recombination. This goal is approached by studying hotspots of recombination, which stimulate a critical, rate-limiting step of recombination. In the bacterium Escherichia coll, studies will focus on Chi hotspots, which stimulate the major (RecBCD) pathway of recombination and DMA break repair. In the fission yeast Schizosaccharomyces pombe, studies will focus on the mutationally created M26 hotspot and the naturally occurring hotspot mbsl. These microbes are especially amenable for genetic and biochemical analyses, but in many ways their recombination mimics that of humans. A unifying theme emerging from the research in E. coll and S. pombe is that homologous recombination is initiated by single-strand and double-strand breaks through the formation of single, not double, Holliday junctions. The specific aims are 1) to elucidate the complex interaction of Chi hotspots and RecBCD enzyme, with special emphasis on testing a specific hypothesis of RecBCD inter-subunit signaling triggered by Chi, 2) to determine the DMA events at mbsl, including a detailed study of novel DMA joint molecule intermediates (single Holliday junctions), and 3) to determine the molecular basis of repression of some but not all M26 hotspot sequences in the genome and thereby further elucidate the genome-wide control of recombination by local chromatin structure. These aims will be achieved by a combination of biochemistry and electron microscopy with purified components, and genetics and DNA analysis 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 DNA double-strand breaks in chromosomes and in the faithful segregation of chromosomes during meiosis. Aberrancies of recombination and DNA break repair are responsible for chromosomal aberrations 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.