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 propose to develop a cell-free system for recombination derived from Escherichia coli. Our substrate for recombination will be DNA from a derivative of phage Lambda that may undergo intramolecular recombination between inverted repeats. Following incubation in an E. coli extract, this DNA will be converted by in vitro packaging into phage which can be assayed by selective plating for parental and recombinant types. Our preliminary investigations and those of others demonstrate that in infected cells this recombination-mediated inversion requires identified E. coli recombinational enzymes - RecA and RecBC - and is stimulated by Chi sites. We will determine the requirement for additional proteins, such as DNA ligase and single-strand binding protein (SSB), and for other processes, such as DNA replication. With the cell-free system, we will identify any additional required components and determine the structures of the DNA intermediates. We will continue our studies of the RecBC enzyme of E. coli to determine the mechanism by which it unwinds and rewinds DNA. We will determine RecBC's of DNA, and its rates of unwinding and rewinding in the presence of other proteins, such as RecA and SSB, known to be involved in recombination. Through a study of the molecular genetics of recBC we will investigate the regulation of expression of the recBC genes, develop a fine structure map of the recBC genes, continue work with Lambda recBC clones isolated in our preliminary work, and seek recBC mutants to evaluate the physiological roles of RecBC enzyme and the mechanisms that regulate its synthesis and activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032194-05
Application #
3280823
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-03-01
Project End
1988-02-29
Budget Start
1987-03-01
Budget End
1988-02-29
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
075524595
City
Seattle
State
WA
Country
United States
Zip Code
98109
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
Nambiar, Mridula; Smith, Gerald R (2018) Pericentromere-Specific Cohesin Complex Prevents Meiotic Pericentric DNA Double-Strand Breaks and Lethal Crossovers. Mol Cell 71:540-553.e4
Ma, Lijuan; Fowler, Kyle R; Martín-Castellanos, Cristina et al. (2017) Functional organization of protein determinants of meiotic DNA break hotspots. Sci Rep 7:1393
Nambiar, Mridula; Smith, Gerald R (2016) Repression of harmful meiotic recombination in centromeric regions. Semin Cell Dev Biol 54:188-97
Polakova, Silvia; Molnarova, Lucia; Hyppa, Randy W et al. (2016) Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation. PLoS Genet 12:e1006102
Ma, Lijuan; Milman, Neta; Nambiar, Mridula et al. (2015) Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair. Nucleic Acids Res 43:7349-59
Phadnis, Naina; Cipak, Lubos; Polakova, Silvia et al. (2015) Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation. PLoS Genet 11:e1005225
Cipak, Lubos; Polakova, Silvia; Hyppa, Randy W et al. (2014) Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase. Nat Protoc 9:223-31
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
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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