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.

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

“…Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20,40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18,34,35).…”

Local regulation of the Srs2 helicase by the SUMO-like domain protein Esc2 promotes recombination at sites of stalled replication

“…7D). STUbLs are known to mediate proteasome degradation of SUMOylated substrates (Sriramachandran and Dohmen 2014), and Srs2 is itself SUMOylated (Saponaro et al 2010). Importantly, here we identified that Esc2 functions upstream of Slx5-Slx8-mediated action as a crucial regulator of Srs2 turnover (Fig.…”

“…The SUMO targeted ubiquitin ligase (STUbL) Slx5-Slx8 complex plays a role in genome stability by controlling the turnover of SUMOylated factors in response to DNA damage (Sriramachandran and Dohmen 2014). As Esc2 and its Schizosaccharomyces pombe ortholog, Rad60, genetically and physically interact with Slx5-Slx8 (Prudden et al 2007;Sollier et al 2009) and as Srs2 is SUMOylated (Saponaro et al 2010;Kolesar et al 2012), we examined whether Srs2 degradation is also mediated by Slx5-Slx8. We found that Srs2 protein levels were stabilized following genotoxic stress in the absence of Slx5, similar to what we observed in esc2Δ (Fig.…”

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…This explains the srs2 Δ puzzling phenotype which includes both recombination deficiency and hyper‐recombination. The anti‐recombination role of Srs2 is particularly important for inhibition of recombination at replication forks and involves complex regulation of Srs2 through post‐translational modifications at its C‐terminus (Saponaro et al , 2010; Kolesar et al , 2012). The C‐terminal part is not needed for the role of Srs2 in re‐synthesis of resected DNA, and therefore, the Srs2 pro‐recombination function is genetically separable from its role at replication forks.…”

“…Loss of Srs2 results in a paradoxical phenotype. On one hand, srs2 mutants are hyper‐recombinogenic (Aguilera & Klein, 1988), and on the other hand, they are deficient in DSB repair via HR and SSA (Vaze et al , 2002; Saponaro et al , 2010). Here we elucidate at the molecular level the role of Srs2 in multiple repair mechanisms involving extended DNA resection by showing that Srs2 is capable of dislodging Rad51 from ssDNA in order to promote loading of proliferating cell nuclear antigen (PCNA) and DNA replication machinery to restore dsDNA at repair loci.…”