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.

Figure 1. Srs2 contains consensus sites for multiple post-translational modifications.

(A) Schematic view of Srs2 with the conserved UvrD helicase domain (light grey box) and the PCNA binding domain that is composed of the last 138 amino acids of the protein (black box). Srs2 contains seven CDK1-dependent consensus sites and, within the PCNA binding domain, three sumoylation consensus sites. (B) Protein extracts were prepared from cultures of SRS2 and srs2 phospho-mutants strains grown in log-phase with or without MMS and analyzed using anti-Srs2 antibodies to assess protein levels and phosphorylation status.

Figure 2. Phosphorylation of Srs2 is required for cell survival when DNA break repair is channeled into HR.

(A) Survival of SRS2, srs2Δ, srs2-7AV, and srs2-7DE strains was determined after exposure to different doses of UV-light and the DSB-inducer zeocin. (B) Tetrads obtained from sporulation of diploids heterozygous for the indicated mutations. Double mutant spores are indicated by the white squares. (C) The indicated strains were grown at an equal cell concentration, sequentially diluted 1:6 and spotted onto plates containing MMS at the indicated concentrations. Cell growth was evaluated after incubation at 28°C for 3 days.

Figure 3. Phosphorylation of Srs2 is required for Rad51-dependent DSB repair via the SDSA pathway.

An HO-mediated DSB was induced by addition of galactose to cultures of SRS2 and srs2 mutants. Cell viability and Rad53 phosphorylation was analyzed during DSB repair by SSA (A) or GC (B). (C) Southern Blot analysis was performed on EcoRI digested DNA extracted from SRS2 and srs2 strains at the indicated time points following galactose induction of the DSB which is repaired by GC. DSB repair efficiency and the percentage of crossover products were calculated at 24 hours after DSB induction.

Figure 4. Srs2 phosphorylation controls turnover of Srs2 but not Rad51 at the invading strand during DSB repair.

Proteins extracts were prepared from the indicated strains, fixed in formaldehyde and analyzed by ChIP using anti-Rad51 (A) or anti-Srs2 antibodies (B). After DNA cross-linking reversion, real-time PCRs were performed to quantitatively analyze the ChIP results. The DNA region amplified by PCR is located on donor sequence on Chromosome III.

Figure 5. Srs2 sumoylation causes recombination defects in the unphosphorylatable srs2-7AV mutant.

(A) Srs2 was immunoprecipitated from protein extracts prepared from the indicated yeast strains under DNA damaging conditions. Blots were probed first with anti-SUMO antibodies, then stripped and reprobed with anti-Srs2 antibodies. (B) Cell survival, DSB repair efficiency and rate of crossover formation were determined for the indicated strains as in Figure 3C. (C) Tetrads obtained from sporulation of diploids heterozygous for the rad27Δ and srs2-7AV3KR mutations. Double mutant spores are indicated by the white squares.

Figure 6. The Cdk1-dependent role of Srs2 does not depend on its interaction with PCNA.

(A) Viability of SRS2 and srs2-7AV strains, with and without the PCNA interacting domain, after DSB induction. (B) Tetrads obtained from sporulation of diploids heterozygous for rad27Δ and the indicated srs2 mutations. Double mutant spores are indicated by the white squares.

Figure 7. Model for the roles of the Srs2 modifications in recombination.

Srs2 removes toxic Rad51 filaments by translocating on ss-DNA. In response to a DSB, Cdk1 targets Srs2 helicase to dismantle the D-loops, thus allowing SDSA pathway that limits DNA exchanges. When Srs2 phosphorylation is prevented, unscheduled sumoylation takes over and the DSB is channeled into the Slx5-dependent pathway.