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Nat Struct Mol Biol. 2014 Apr;21(4):405-12. doi: 10.1038/nsmb.2786. Epub 2014 Mar 9.

RPA antagonizes microhomology-mediated repair of DNA double-strand breaks.

Author information

1
Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, New York, New York, USA.
2
Department of Biochemistry and Biophysics, Columbia University College of Physicians and Surgeons, New York, New York, USA.
3
1] Department of Biochemistry and Biophysics, Columbia University College of Physicians and Surgeons, New York, New York, USA. [2] Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, New York, New York, USA.

Abstract

Microhomology-mediated end joining (MMEJ) is a Ku- and ligase IV-independent mechanism for the repair of DNA double-strand breaks that contributes to chromosome rearrangements. Here we used a chromosomal end-joining assay to determine the genetic requirements for MMEJ in Saccharomyces cerevisiae. We found that end resection influences the ability to expose microhomologies; however, it is not rate limiting for MMEJ in wild-type cells. The frequency of MMEJ increased by up to 350-fold in rfa1 hypomorphic mutants, suggesting that replication protein A (RPA) bound to the single-stranded DNA (ssDNA) overhangs formed by resection prevents spontaneous annealing between microhomologies. In vitro, the mutant RPA complexes were unable to fully extend ssDNA and were compromised in their ability to prevent spontaneous annealing. We propose that the helix-destabilizing activity of RPA channels ssDNA intermediates from mutagenic MMEJ to error-free homologous recombination, thus preserving genome integrity.

Comment in

PMID:
24608368
PMCID:
PMC3980576
DOI:
10.1038/nsmb.2786
[Indexed for MEDLINE]
Free PMC Article

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