The broad objective of this proposal is to understand the biochemical events that comprise the process of genetic recombination. This goal will be achieved by reconstituting various steps of the recombination process in vitro, using purified proteins from Escherichia coli and from Saccharomyces cerevisiae, and also by understanding the mechanism of these molecular events using defined biochemical reactions. Genetic recombination is a fundamental biological process that involves the processing of broken DNA, homologous recognition, exchange of DNA strands, and resolution of the recombination intermediates. It is an important cellular process that is used by all organisms to repair DNA damage, restart DNA replication, and generate genetic diversity. Two specific objectives are planned. The first set of aims is to fully reconstitute in vitro the genetic recombination process of E. coli. The second major aim is to determine the biochemical mechanism of steps that define recombination in the eukaryote, S. cerevisiae. Understanding the mechanism of recombination will provide insight into the manner by which DNA breaks are repaired and, thereby, ensure that genomes are accurately repaired and maintained. Failure to repair DNA properly can ultimately lead to cancer development. The human homologs of some of the proteins that we will investigate include the Breast Cancer Susceptibility Gene 2 (BRCA2) and the Bloom (BLM) helicase. Understanding the mechanism of recombination should also provide insight into possible new experimental approaches for gene replacement therapies.

Public Health Relevance

Recombination is an important cellular process that is used by all organisms to repair DNA damage. Understanding the mechanism of recombination will provide insight into the manner by which DNA breaks are repaired and, thereby, ensure that genomes are accurately repaired and maintained. Failure to repair DNA properly can ultimately lead to cancer development, premature aging, and anemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062653-33
Application #
8447533
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Janes, Daniel E
Project Start
1982-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
33
Fiscal Year
2013
Total Cost
$509,683
Indirect Cost
$174,349
Name
University of California Davis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Bocquet, Nicolas; Bizard, Anna H; Abdulrahman, Wassim et al. (2014) Structural and mechanistic insight into Holliday-junction dissolution by topoisomerase III? and RMI1. Nat Struct Mol Biol 21:261-8
Cannavo, Elda; Cejka, Petr; Kowalczykowski, Stephen C (2013) Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection. Proc Natl Acad Sci U S A 110:E1661-8
Jensen, Ryan B; Ozes, Ali; Kim, Taeho et al. (2013) BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage. DNA Repair (Amst) 12:306-11
Forget, Anthony L; Dombrowski, Christopher C; Amitani, Ichiro et al. (2013) Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy. Nat Protoc 8:525-38
Nimonkar, Amitabh V; Genschel, Jochen; Kinoshita, Eri et al. (2011) BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair. Genes Dev 25:350-62
Forget, Anthony L; Kowalczykowski, Stephen C (2010) Single-molecule imaging brings Rad51 nucleoprotein filaments into focus. Trends Cell Biol 20:269-76
Hilario, Jovencio; Kowalczykowski, Stephen C (2010) Visualizing protein-DNA interactions at the single-molecule level. Curr Opin Chem Biol 14:15-22
Cejka, Petr; Cannavo, Elda; Polaczek, Piotr et al. (2010) DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2. Nature 467:112-6
Cejka, Petr; Plank, Jody L; Bachrati, Csanad Z et al. (2010) Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nat Struct Mol Biol 17:1377-82
Amitani, Ichiro; Liu, Bian; Dombrowski, Christopher C et al. (2010) Watching individual proteins acting on single molecules of DNA. Methods Enzymol 472:261-91

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