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

• Published in: Nature structural & molecular biology Vol. 21; no. 4; pp. 405 - 412
• Main Authors: Deng, Sarah K, Gibb, Bryan, de Almeida, Mariana Justino, Greene, Eric C, Symington, Lorraine S
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.04.2014; Nature Publishing Group
• Subjects: 45; 45/70; 631/337/149; Bioassays; Biochemistry; Biological Microscopy; Brewer's yeast; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA repair; DNA, Single-Stranded - metabolism; Genetic research; Genetics; Homologous Recombination; Life Sciences; Membrane Biology; Mutagenesis; Mutants; Protein research; Protein Structure; Replication Protein A - genetics; Replication Protein A - metabolism; Replication Protein A - physiology; RNA Polymerase I - genetics; RNA Polymerase I - metabolism; RNA Polymerase I - physiology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Saccharomyces cerevisiae Proteins - physiology; Structure
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb.2786

Resection of DNA double strand–break ends generates single strands that can spontaneously anneal to undergo mutagenic microhomology-mediated end joining (MMEJ). A combination of genetic and biophysical assays now shows that replication protein A (RPA) thwarts strand annealing by binding to the resected ends to promote Rad51 filament assembly and error-free repair by homologous recombination. 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.