Role of the Rad52 amino-terminal DNA binding activity in DNA strand capture in homologous recombination

J Biol Chem. 2009 Nov 27;284(48):33275-84. doi: 10.1074/jbc.M109.057752. Epub 2009 Oct 6.

Abstract

Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised amino-terminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that rad52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type Rad52. Analysis of gene conversion intermediates reveals that rad52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of Rad52 is needed for the capture of the second DNA end during homologous recombination.

Publication types

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

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Chromatin Immunoprecipitation
  • DNA Breaks, Double-Stranded
  • DNA Repair*
  • DNA, Single-Stranded
  • Electrophoresis, Polyacrylamide Gel
  • Microscopy, Electron
  • Mutation
  • Protein Binding
  • Rad51 Recombinase / genetics
  • Rad51 Recombinase / metabolism
  • Rad52 DNA Repair and Recombination Protein / chemistry
  • Rad52 DNA Repair and Recombination Protein / genetics
  • Rad52 DNA Repair and Recombination Protein / metabolism*
  • Recombination, Genetic*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*

Substances

  • DNA, Single-Stranded
  • Rad52 DNA Repair and Recombination Protein
  • Saccharomyces cerevisiae Proteins
  • Rad51 Recombinase