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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair*
  • DNA, Single-Stranded / metabolism
  • Homologous Recombination
  • RNA Polymerase I / genetics
  • RNA Polymerase I / metabolism
  • RNA Polymerase I / physiology
  • Replication Protein A / genetics
  • Replication Protein A / metabolism
  • Replication Protein A / physiology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / physiology*


  • DNA, Single-Stranded
  • RFA1 protein, S cerevisiae
  • RFA2 protein, S cerevisiae
  • RPA14 protein, S cerevisiae
  • Replication Protein A
  • Saccharomyces cerevisiae Proteins
  • RNA Polymerase I