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Proc Natl Acad Sci U S A. 2015 Dec 15;112(50):E6907-16. doi: 10.1073/pnas.1507833112. Epub 2015 Nov 25.

DNA polymerases δ and λ cooperate in repairing double-strand breaks by microhomology-mediated end-joining in Saccharomyces cerevisiae.

Author information

1
Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665;
2
Department of Microbiology and Molecular Genetics, University of California, Davis, CA 95616-8665; Department of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665 wdheyer@ucdavis.edu.

Abstract

Maintenance of genome stability is carried out by a suite of DNA repair pathways that ensure the repair of damaged DNA and faithful replication of the genome. Of particular importance are the repair pathways, which respond to DNA double-strand breaks (DSBs), and how the efficiency of repair is influenced by sequence homology. In this study, we developed a genetic assay in diploid Saccharomyces cerevisiae cells to analyze DSBs requiring microhomologies for repair, known as microhomology-mediated end-joining (MMEJ). MMEJ repair efficiency increased concomitant with microhomology length and decreased upon introduction of mismatches. The central proteins in homologous recombination (HR), Rad52 and Rad51, suppressed MMEJ in this system, suggesting a competition between HR and MMEJ for the repair of a DSB. Importantly, we found that DNA polymerase delta (Pol δ) is critical for MMEJ, independent of microhomology length and base-pairing continuity. MMEJ recombinants showed evidence that Pol δ proofreading function is active during MMEJ-mediated DSB repair. Furthermore, mutations in Pol δ and DNA polymerase 4 (Pol λ), the DNA polymerase previously implicated in MMEJ, cause a synergistic decrease in MMEJ repair. Pol λ showed faster kinetics associating with MMEJ substrates following DSB induction than Pol δ. The association of Pol δ depended on RAD1, which encodes the flap endonuclease needed to cleave MMEJ intermediates before DNA synthesis. Moreover, Pol δ recruitment was diminished in cells lacking Pol λ. These data suggest cooperative involvement of both polymerases in MMEJ.

KEYWORDS:

DNA repair; genome stability; translocation

PMID:
26607450
PMCID:
PMC4687552
DOI:
10.1073/pnas.1507833112
[Indexed for MEDLINE]
Free PMC Article

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