The long-term goal of the proposed research is to elucidate 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 the mutationally created M26 hotspot and on mbs1 and other naturally occurring hotspots. 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 a specific hypothesis of RecBCD inter-subunit signaling triggered by Chi, 2) to determine the proteins required for formation and resolution of joint molecules, including novel single (rather than double) Holliday junctions, and to compare different hotspots in this respect, and 3) to determine the roles of chromatin modifications and chromosomal proteins in regulating hotspots throughout the genome.
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.

Public Health Relevance

Genetic recombination is important for the repair of broken DNA and for the proper segregation of chromosomes during meiosis (sex-cell formation). Faulty recombination can result in cancer cells or birth defects. By studying hotspots of recombination we shall expand our knowledge of the molecular basis of this process important for human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031693-29
Application #
8105506
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
1982-07-01
Project End
2012-07-31
Budget Start
2011-08-01
Budget End
2012-07-31
Support Year
29
Fiscal Year
2011
Total Cost
$742,797
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
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
Martín-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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