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.
|Gong, Yi; Handa, Naofumi; Kowalczykowski, Stephen C et al. (2017) PHF11 promotes DSB resection, ATR signaling, and HR. Genes Dev 31:46-58|
|Martinez, Juan S; von Nicolai, Catharina; Kim, Taeho et al. (2016) BRCA2 regulates DMC1-mediated recombination through the BRC repeats. Proc Natl Acad Sci U S A 113:3515-20|
|Bell, Jason C; Kowalczykowski, Stephen C (2016) RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci 41:491-507|
|Pavankumar, T L; Exell, J C; Kowalczykowski, S C (2016) Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases. Methods Enzymol 581:1-32|
|Chen, Huan; Donnianni, Roberto A; Handa, Naofumi et al. (2015) Sae2 promotes DNA damage resistance by removing the Mre11-Rad50-Xrs2 complex from DNA and attenuating Rad53 signaling. Proc Natl Acad Sci U S A 112:E1880-7|
|Bell, Jason C; Liu, Bian; Kowalczykowski, Stephen C (2015) Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function. Elife 4:e08646|
|Wang, Anderson T; Kim, Taeho; Wagner, John E et al. (2015) A Dominant Mutation in Human RAD51 Reveals Its Function in DNA Interstrand Crosslink Repair Independent of Homologous Recombination. Mol Cell 59:478-90|
|Kowalczykowski, Stephen C (2015) An Overview of the Molecular Mechanisms of Recombinational DNA Repair. Cold Spring Harb Perspect Biol 7:|
|Fasching, Clare L; Cejka, Petr; Kowalczykowski, Stephen C et al. (2015) Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism. Mol Cell 57:595-606|
|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|
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