Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2

Cell Rep. 2017 Dec 12;21(11):3166-3177. doi: 10.1016/j.celrep.2017.11.047.

Abstract

Srs2 is a superfamily 1 (SF1) helicase and antirecombinase that is required for genome integrity. However, the mechanisms that regulate Srs2 remain poorly understood. Here, we visualize Srs2 as it acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. We demonstrate that Srs2 is a processive translocase capable of stripping thousands of Rad51 molecules from ssDNA at a rate of ∼50 monomers/s. We show that Srs2 is recruited to RPA clusters embedded between Rad51 filaments and that multimeric arrays of Srs2 assemble during translocation on ssDNA through a mechanism involving iterative Srs2 loading events at sites cleared of Rad51. We also demonstrate that Srs2 acts on heteroduplex DNA joints through two alternative pathways, both of which result in rapid disruption of the heteroduplex intermediate. On the basis of these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.

Keywords: DNA curtains; DNA repair; Rad51; Srs2; helicase; homologous recombination; single molecule.

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • DNA Helicases / genetics*
  • DNA Helicases / metabolism
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • DNA, Fungal / genetics
  • DNA, Fungal / metabolism
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Gene Expression Regulation, Fungal*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Homologous Recombination*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mutation
  • Nucleic Acid Heteroduplexes / genetics*
  • Nucleic Acid Heteroduplexes / metabolism
  • Protein Binding
  • Rad51 Recombinase / genetics*
  • Rad51 Recombinase / metabolism
  • Red Fluorescent Protein
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • DNA, Fungal
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Luminescent Proteins
  • Nucleic Acid Heteroduplexes
  • RAD55 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • SRS2 protein, S cerevisiae
  • Green Fluorescent Proteins
  • RAD51 protein, S cerevisiae
  • Rad51 Recombinase
  • Adenosine Triphosphatases
  • RAD57 protein, S cerevisiae
  • DNA Helicases
  • DNA Repair Enzymes