Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair

PLoS Genet. 2010 Feb 26;6(2):e1000858. doi: 10.1371/journal.pgen.1000858.

Abstract

Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.

Publication types

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

MeSH terms

  • CDC28 Protein Kinase, S cerevisiae / metabolism*
  • Consensus Sequence
  • DNA Breaks, Double-Stranded
  • DNA Helicases / chemistry
  • DNA Helicases / metabolism*
  • DNA Repair*
  • DNA, Fungal / biosynthesis*
  • Microbial Viability
  • Models, Biological
  • Mutation / genetics
  • Phosphorylation
  • Proliferating Cell Nuclear Antigen / metabolism
  • Protein Binding
  • Protein Processing, Post-Translational
  • Rad51 Recombinase / metabolism
  • Recombination, Genetic*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Small Ubiquitin-Related Modifier Proteins / metabolism

Substances

  • DNA, Fungal
  • Proliferating Cell Nuclear Antigen
  • Saccharomyces cerevisiae Proteins
  • Small Ubiquitin-Related Modifier Proteins
  • SRS2 protein, S cerevisiae
  • CDC28 Protein Kinase, S cerevisiae
  • Rad51 Recombinase
  • DNA Helicases