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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Portnoy, Matthew
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Fred Hutchinson Cancer Research Center
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Fowler, Kyle R; Hyppa, Randy W; Cromie, Gareth A et al. (2018) Physical basis for long-distance communication along meiotic chromosomes. Proc Natl Acad Sci U S A 115:E9333-E9342
Amundsen, Susan K; Smith, Gerald R (2018) The RecB helicase-nuclease tether mediates Chi hotspot control of RecBCD enzyme. Nucleic Acids Res :
Nuckolls, Nicole L; Bravo Núñez, María Angélica; Eickbush, Michael T et al. (2017) wtf genes are prolific dual poison-antidote meiotic drivers. Elife 6:
Nambiar, Mridula; Smith, Gerald R (2016) Repression of harmful meiotic recombination in centromeric regions. Semin Cell Dev Biol 54:188-97
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
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
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
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
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

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