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

Sarah K Deng1, Bryan Gibb2, Mariana Justino de Almeida1

  • 1Department of Microbiology and Immunology, Columbia University College of Physicians and Surgeons, New York, New York, USA.

Nature Structural & Molecular Biology
|March 11, 2014
PubMed

Insights

Replication protein A (RPA) prevents mutagenic DNA repair by limiting microhomology-mediated end joining (MMEJ). This finding reveals RPA

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Microhomology-mediated end joining (MMEJ) repairs DNA double-strand breaks and can cause chromosome rearrangements.
  • MMEJ is independent of Ku and ligase IV, key proteins in other DNA repair pathways.

Purpose of the Study:

  • To investigate the genetic factors influencing MMEJ in Saccharomyces cerevisiae.
  • To elucidate the role of replication protein A (RPA) in regulating MMEJ.

Main Methods:

  • Utilized a chromosomal end-joining assay in Saccharomyces cerevisiae.
  • Analyzed MMEJ frequency in wild-type and rfa1 hypomorphic mutants.
  • Performed in vitro assays with mutant RPA complexes.

Main Results:

  • End resection exposes microhomologies but is not rate-limiting for MMEJ in wild-type cells.
  • MMEJ frequency increased up to 350-fold in rfa1 mutants.
  • Mutant RPA complexes showed defects in single-stranded DNA (ssDNA) extension and annealing prevention.

Conclusions:

  • RPA bound to ssDNA overhangs inhibits spontaneous microhomology annealing, preventing mutagenic MMEJ.
  • RPA's helix-destabilizing activity may direct ssDNA intermediates towards error-free homologous recombination.
  • This mechanism is crucial for maintaining genome integrity.

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