Cdk1 Targets Srs2 to Complete Synthesis-Dependent Strand Annealing and to Promote Recombinational Repair

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. Broken DNA molecules can be repaired by copying a homologous DNA sequence located elsewhere in the genome. This process, called homologous recombination, needs to be carefully regulated, because unwanted DNA exchanges can lead to genome rearrangements and cell death. Cdk1 kinase is required for cell cycle progression and phosphorylates DNA repair factors, such as Srs2, a protein that can both translocate on single-stranded DNA and open the two strands of DNA double helix. DNA translocation activity of Srs2 is crucial to prevent unwanted recombination, while DNA unwinding activity might be important to promote recombination. In this study, we used two srs2 mutants that constitutively express the unphosphorylated or Cdk1-dependent phosphorylated Srs2 protein isoforms. We found that Srs2 performs genetically distinct functions in preventing or promoting homologous recombination. Cdk1 targets Srs2 to promote accurate repair of double-stranded DNA breaks, but is not essential for the removal of toxic recombination intermediates assembled at single-stranded DNA breaks. Further, Cdk1 counteracts sumoylation of Srs2, which is responsible for recombination defects due to the lack of Srs2 phosphorylation. In summary, Cdk1-dependent Srs2 phosphorylation prevents its unscheduled sumoylation and targets the helicase to promote accurate homologous recombinational repair.