The long-term objective of the research proposed here is to determine the mechanism of generalized genetic recombination in terms of the enzymes promoting the individual steps of this process and the structures of the DNA intermediates at each step. An additional objective is to determine the mechanisms by which recombination is regulated. We will continue our studies of the Escherichia coli RecBCD enzyme, which is required for the principal pathway of recombination in E. coli. We will determine the mechanisms by which this enzyme unwinds and rewinds DNA and by which it hydrolyzes DNA. We will couple the activities of RecBCD enzyme and RecA and SSB proteins to produce recombinational intermediates from duplex DNA substrates. We will determine the ability of RecBCD enzyme to cleave D-loops and to resolve Holliday junctions, two postulated intermediates in recombination. We will isolate and study mutants differentially altered in the multiple activities of RecBCD enzyme to elucidate their roles in recombination and other cellular processes (recovery from DNA damage and maintenance of cell viability). We will continue our studies of the regulation of expression of the recBCD genes, to elucidate a mechanism by which recombination may be controlled. To compare the mechanisms of recombination in organisms other than E. coli, we will clone the genes coding for RecBCD-like enzymes of other bacteria and determine the ability of these enzymes to promote recombination in E. coli and in the original bacteria. We will investigate the activities of these enzymes on DNA. We will search for recombination-related enzymatic activities in the fission yeast Schizosaccharomyces pombe. We will isolate S. pombe mutants altered in recombination or lacking relevant enzymatic activities. These mutants and enzymes will provide a foundation for studying the molecular mechanisms of recombination in eukaryotes and for comparing these mechanisms with those in prokaryotes. Recombination is important for generating diversity at both the organismal and cellular levels. Aberrancies of recombination generate chromosomal rearrangements, such as deletions and translocations. Chromosomal rearrangements are associated with, and may be a cause of, birth defects and cancers. Understanding the molecular mechanisms of recombination is important in determining the causes of and possibly preventing these diseases.
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