Abstract

The research proposed here is directed toward an understanding of the molecular mechanism of general genetic recombination. This goal is approached through the study of special sites in DNA promoting a high rate of recombination in their vicinity and through the identification and study of the activity of proteins interacting with these sites. The Chi recombinational hotspots of bacteriophage lambda and its host Escherichia coli, and the M26 recombinational hotspot of the fission yeast Schizosaccharomyces pombe will be studied in the proposed research. In addition, the interaction of Chi and RecBCD enzyme, which directly activates Chi, site will be sought. These studies will elucidated the molecular mechanisms by which recombinational hotspots act and , hence, elucidate an important (rate-limiting) step in homologous recombination.
Specific aims are to: (1) Determine if RecBCD enzyme is altered after encountering Chi. (2) Couple the production of ssDNA ends at Chi by RecBCD enzyme to the production of hybrid DNA by RecA and SSB proteins. (3) Locate with high resolution the point of Chi-stimulated genetic exchange, and determine whether the action of cellular enzymes, such as nucleases, alters this point. (4) Determine whether Chi acts at an early or late step of recombination. (5) Search for Chi-dependent DNA strand cleavage by RecBCD enzyme in E. coli cells. (6) Determine the basis of the context- dependence of the M26 hotspot activity in S. pombe. (7) Isolate mutants of S. pombe that fail to activate the M26 hotspot, or that activate other nucleotide sequences as hotspots. (8) Analyze S. pombe rec mutants for locus specificity. Recombination plays an important role in generating diversity among individuals in a population and, in some cases, among cells within an organism. Understanding the mechanism of recombination thus lends insight into the evolution and development of organisms. Aberrations of recombination may be responsible for certain chromosomal rearrangements associated with birth defects and cancer.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031693-09
Application #
3279935
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1982-07-01
Project End
1994-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109

  Publications

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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