Project Description The broad objective of this proposal is to understand the mechanism of genetic recombination and its relationship to chromosome maintenance in both Bacteria and Eukarya. Our approach is to reconstitute increasingly more complex reactions that recapitulate steps of the recombination process, using purified proteins from Escherichia coli and from Saccharomyces cerevisiae, and using sophisticated biochemical and biophysical methods of analysis, which include single-molecule imaging.
The specific aims of this proposal are divided along two broad aims. The first set of aims describes our ongoing efforts to mechanistically understand the steps of E. coli recombination in vitro.
This aim i ncludes studies on the function of RecN protein;the mechanism of the DNA homology search by RecA;and the processing of double Holliday junctions. The second major aim is to continue to biochemically reconstitute the processes that comprise recombination in S. cerevisiae.
This aim i ncludes studies of the function of the chromatin remodelers;the coupling of DNA resection to DNA pairing;and the mechanism of the DNA homology search by Rad51.
The broad objective of this proposal is to understand the mechanism of genetic recombination and its relationship to chromosome maintenance in both Bacteria and Eukarya. In doing so, we hope to understand universal principles that underpin the molecular events which comprise recombinational DNA repair. Mutations in many human analogs or orthologs of the proteins being studies here (e.g., BLM, BRCA2, MRN complex, and the RAD51 paralogs) give rise to defective recombinational DNA repair, chromosomal instability, and predisposition to cancers, as well as to hematological malignancies, bone marrow failure, and developmental abnormalities in Fanconi's anemia. We expect that our experiments will continue to both inform and provide new insight into how these proteins cooperate to repair broken DNA.
|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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