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 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 the M26 hotspot and the recently discovered hotspot mbsl, both of which stimulate meiotic recombination through the formation of DNA double-strand breaks. A unifying theme emerging from the research in E. coli and S. pombe is that homologous recombination can frequently occur far from broken DNA ends.
The specific aims are 1) to elucidate the complex interaction of Chi hotspots and RecBCD enzyme, with special emphasis on the interdependencies of the multiple enzymatic and physical changes of RecBCD enzyme and DNA resulting from this interaction, 2) to determine the basis of the context-dependence of M26 hotspot activity and to assess the role of M26-1ike sites in wild-type S. pombe, and 3) to investigate the mbsl hotspot, with special emphasis on testing the hypothesis that meiotic gene conversion and crossing-over can be separated by a long distance (tens of kilobases).
These aims will be achieved by a combination of biochemistry and electron microscopy with purified components, and genetics with intact cells. The results of these studies will elucidate the regulation of recombination both spatially along chromosomes and temporally 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 rearrangements associated with cancer, birth defects, and certain hereditary diseases. Gene therapy by homologous gene replacement and gene targeting to generate experimental animals will be facilitated by understanding the molecular mechanism of homologous recombination and its stimulation by hotspots.
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