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, and this proposal specifically focuses on two aspects of HR that are woefully understudied: gap repair and DNA synthesis. DNA polymerases are a well-studied class of enzymes, but their specific functions in HR are unknown. Gaps in DNA can result from stalling of replication forks, and aberrant recovery of compromised forks leads to gross chromosomal rearrangements, a hallmark of cancer cells.
The Specific Aims are: (1) Determine the mechanistic and biological consequences of the Rad54-PCNA interaction. The Rad54 motor protein plays a key role in HR, dissociating Rad51 from heteroduplex DNA to allow DNA polymerases access to the invading 3'end. We have identified a PCNA interaction site on the surface of Rad54 projecting towards the DNA axis, and we expect that this exciting finding will help to reveal the mechanisms involved in the transition from DNA strand invasion to DNA synthesis during HR. (2) Determine the identities of DNA polymerases that are involved in HR. The astounding number of nuclear DNA polymerases in eukaryotes makes yeast the ideal model system to unravel the complexity of DNA synthesis during HR. We will integrate in vivo and in vitro approaches to identify which DNA polymerases and cofactors are involved in HR. In reconstituted recombination reactions, we will determine the specific functions of individual DNA polymerases and cofactors. A key question is whether translesion DNA polymerases are directly involved in HR. (3) Determine the mechanisms of recombination in gap repair. Recombinational repair of DNA gaps is a key feature of replication fork support, but little is known about the specific intermediates or mechanisms involved. Paranemic joints (DNA joints without true intertwining of DNA strands) are an obligatory intermediate in HR-mediated gap repair. Integrating in vitro and in vivo approaches, we will test the requirements for paranemic joint formation and the novel model that the DNA structure- selective endonucleas Mus81-Mms4 specifically cleaves paranemic joints.

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 double-strand break 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)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058015-12
Application #
8304936
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Janes, Daniel E
Project Start
2000-01-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
12
Fiscal Year
2012
Total Cost
$347,794
Indirect Cost
$115,020
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
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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