Distinct Cdk1 Requirements during Single-Strand Annealing, Noncrossover, and Crossover Recombination

Abstract

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in haploid cells is generally restricted to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. This cell cycle specificity depends on cyclin-dependent protein kinases (Cdk1 in Saccharomyces cerevisiae), which initiate HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps is unknown. Here we show that yku70Δ cells, which accumulate single-stranded DNA (ssDNA) at the DSB ends independently of Cdk1 activity, are able to repair a DSB by single-strand annealing (SSA) in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by single-strand annealing and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes.

WEE1 murine deficiency induces hyper-activation of APC/C and results in genomic instability and carcinogenesis

“…39 First, the levels of these mitotic proteins were affected by WEE1 in unsynchronized cells, where most cells are in G1 phase and the CDK1 activity is low (Figures 2a, c, d and e, and Supplementary Figures 1b and c). 40,41 Moreover, levels of mitotic regulators were changed both under conditions that CDK1 phosphorylation was reduced after Wee1 deletion (Figure 2a and Supplementary Figure 1a) and under conditions where CDK1 was fully phosphorylated on Tyr-15 and no difference in phosphorylation was observed after over-expressing WEE1 (Figures 2d and e and Supplementary Figures 1d and e). More importantly, treatment with a CDK1 inhibitor, R0-3306, failed to rescue the defect in the levels of APC/C targets after deletion of Wee1 (Supplementary Figures 1f and h), suggesting that other WEE1 targets may mediate, at least in some part, this response.…”

DNA repair choice defines a common pathway for recruitment of chromatin regulators

“…In addition, loss of Ku70 allows Rad52- and Rad51-dependent recombination events to occur efficiently in G1 arrested cells 30 . To test whether the decreased recruitment of chromatin factors in G1 is due to limited DSB processing, an yku70Δ strain was arrested in G1, and ChIP assays were performed at an HO-induced DSB.…”

Mechanism and regulation of DNA end resection in eukaryotes

“…Inhibition of CDK in yku80Δ (Ku is encoded by YKU70 and YKU80 in yeast) G1 or G2-M arrested cells prevents long-range resection, but not short-range resection suggesting Ku controls short-range resection by modulating MRX and Exo1 access to ends, whereas high CDK activity is required for resection initiation when Ku is present as well as for extensive end processing (Clerici et al, 2008). In contrast to yku70Δ , the rad9Δ mutant does not exhibit increased resection in G1-arrested cells; however, in the yku70Δ rad9Δ double mutant both initiation and extensive resection can occur in G1-arrested cells (Trovesi et al, 2011). Since Dna2 is not active in G1 cells (see below), resection under these circumstances is most likely due to Exo1 activity.…”

“…Removal of Lig4 also increased HR, but not to the same extent as Ku, in agreement with the dual role of Ku in suppressing end resection and promoting NHEJ (Pierce et al, 2001). Even in G1-arrested yeast cells, Rad51-dependent gene conversion between ectopic repeats can be restored in the absence of Ku (Trovesi et al, 2011). Interestingly, crossovers were not detected in G1 yku70Δ cells, indicating an additional layer of regulation by CDK beyond end resection.…”