Double-stranded DNA breaks (DSBs) are the primary genotoxic lesion of ionizing radiation (IR). DSB induction appears to determine the efficacy of IR and other DNA metabolism-based anti-tumor drugs as cancer therapeutic agents. Homologous recombination (HR) is an important DSB repair pathway and essential for cellular radiation resistance and genome stability. HR defects lead to genomic instability, a hallmark in the etiology of cancer. The long-term goal is to elucidate the mechanisms of HR. We focus on the postsynaptic steps of DNA synthesis that determine fidelity of recombinational DNA repair.
The Specific Aims are: (1) Determine the mechanisms of recombination-associated DNA synthesis. Yeast is an ideal model system to unravel the complexity of DNA synthesis during HR. Distinct DNA synthesis steps are invoked in recombination models, and at least four different DNA polymerases have already been implicated. We will delineate the precise role of DNA Pol? in HR using biochemical and molecular approaches (Subaim 1A). We have identified a novel role of Pol 4 in homology-directed repair and will define its specific function by a combination of biochemical and genetic approaches (Subaim 1C). The experiments will also address the key question whether TLS polymerases are directly involved in recombination-associated DNA synthesis. In Subaims 1B and D, we will validate the general significance of our findings using the well conserved human proteins. (2) Identify mechanisms for regulating recombination-associated DNA synthesis. Multiple lines of genetic evidence from several model systems suggest that recombination-associated DNA synthesis is regulated but the mechanisms involved are unknown. We have identified a novel role of the Sgs1-Top3-Rmi1 (human BLM- TOPO3?-RMI1/2) to specifically dissolve yeast Rad51-mediated D-loops. Contrary to expectation this function is independent of Sgs1 helicase activity. We will define this novel mechanism in Subaim 2A and determine whether it cooperates with mismatch repair to proofread recombination and reject strand invasions with mismatches in Subaim 2B. The novel paradigms will be validated in Subaim 2C with the corresponding and highly conserved human proteins.

Public Health Relevance

Homologous recombination is a key DNA repair pathway for DNA double-stranded breaks and other types of complex DNA damage. DNA double-stranded breaks are the critical lesion induced by ionizing radiation and other modalities in cancer treatment. The work in this proposal will lead to an improved mechanistic understanding of recombinational DNA repair, which is fundamental in using biological approaches to improve the efficacy and reduce the side-effects of DNA damage-based anti-tumor therapy.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM058015-13A1
Application #
8758698
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Janes, Daniel E
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California Davis
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Davis
State
CA
Country
United States
Zip Code
95618
Mukherjee, Sucheta; Wright, William Douglass; Ehmsen, Kirk Tevebaugh et al. (2014) The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage. Nucleic Acids Res 42:6511-22
Wright, William Douglass; Heyer, Wolf-Dietrich (2014) Rad54 functions as a heteroduplex DNA pump modulated by its DNA substrates and Rad51 during D loop formation. Mol Cell 53:420-32
Zinovyev, Andrei; Kuperstein, Inna; Barillot, Emmanuel et al. (2013) Synthetic lethality between gene defects affecting a single non-essential molecular pathway with reversible steps. PLoS Comput Biol 9:e1003016
Sneeden, Jessica L; Grossi, Sara M; Tappin, Inger et al. (2013) Reconstitution of recombination-associated DNA synthesis with human proteins. Nucleic Acids Res 41:4913-25
Munoz-Galvan, Sandra; Tous, Cristina; Blanco, Miguel G et al. (2012) Distinct roles of Mus81, Yen1, Slx1-Slx4, and Rad1 nucleases in the repair of replication-born double-strand breaks by sister chromatid exchange. Mol Cell Biol 32:1592-603
Zhang, Xiao-Ping; Heyer, Wolf-Dietrich (2011) Quality control of purified proteins involved in homologous recombination. Methods Mol Biol 745:329-43
Wright, William D; Ehmsen, Kirk T; Heyer, Wolf-Dietrich (2011) Assays for structure-selective DNA endonucleases. Methods Mol Biol 745:345-62
Ceballos, Shannon J; Heyer, Wolf-Dietrich (2011) Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination. Biochim Biophys Acta 1809:509-23
Schwartz, Erin K; Heyer, Wolf-Dietrich (2011) Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes. Chromosoma 120:109-27
Liu, Jie; Heyer, Wolf-Dietrich (2011) Who's who in human recombination: BRCA2 and RAD52. Proc Natl Acad Sci U S A 108:441-2

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