Regulation of the DNA damage response by cyclin-dependent kinase in "Saccharomyces cerevisiae ”

The eukaryotic cell division cycle comprises a series of events, whose ordering and correct progression depends on the oscillating activity of cyclin-dependent kinases (Cdks), which safeguard timely duplication and segregation of the genome. Since genome integrity is constantly threatened by both endogenous and exogenous sources of DNA damage, cell division cycle is intimately connected to an evolutionarily conserved DNA damage response (DDR) in order to guarantee the faithful transmission of genetic information from one cell to its daughter and to ensure cell survival. In fact, the DDR involves DNA repair pathways that reverse DNA lesions, as well as DNA damage checkpoint pathways that inhibit cell-cycle progression while repair occurs. The DNA damage checkpoint is activated in the presence of DNA damage or replicative stress and is based on signal transduction cascades of protein kinases that recognize damaged DNA, transduce and amplify the damage signal, and target several effector proteins to prevent cell cycle progression and to couple it with the DNA repair capacity. In addition to driving cell-cycle arrest, these pathways control the activation of DNA repair pathways, the proper completion of DNA replication, the activation of transcriptional programs and, in some cases, the commitment to cell death by apoptosis. There is increasing evidence that Cdks are not only downstream targets of the DDR, but also participate in the DDR regulation, both by leading to a strong checkpoint activation and by promoting DNA repair by homologous recombination (HR). The most dangerous DNA lesions are the DNA double-strand breaks (DSBs) that can be repaired either by non-homologous end joining (NHEJ) or by HR. While NHEJ directly relegates the broken DNA ends, HR uses homologous DNA sequences as a template to form recombinants that are either crossover or noncrossover with regard to flanking parental sequences. Furthermore, a DSB flanked by direct DNA repeats can be repaired by a particular HR pathway called single-strand annealing (SSA), which results in DSB repair with concomitant deletion of one repeat and of the intervening sequence. All HR processes initiate with extensive 5’ to 3’ end-processing (a process referred to as 5’-3’ resection) of the broken ends to yield 3’-ended single-stranded DNA (ssDNA) tails, which are bound by Replication Protein A (RPA). RPA is then displaced by Rad51 to form nucleoprotein filaments that can catalyse homologous pairing and strand invasion. The choice between NHEJ and HR pathways is tightly regulated during the cell cycle and HR 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 Cdk (Cdk1 in Saccharomyces cerevisiae) activity, which initiates HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps was unknown. To address this question, we explored the Cdk1 requirement in the execution of different HR processes in S. cerevisiae. In order to bypass the Cdk1 requirement for resection we analyzed cells lacking Yku heterodimer and/or the checkpoint protein Rad9, which are known as negative regulators of DSB resection. We showed that yku70Δ cells, which accumulate ssDNA at the DSB ends independently of Cdk1 activity, are able to repair a DSB by 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 SSA and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes. As crossovers during mitotic cell growth have the potential for deleterious genome rearrangements when the sister chromatid is not used as repair template, this additional function of Cdk1 in promoting crossovers can provide another safety mechanism to ensure genome stability. During DNA replication cells are particularly sensitive to DNA damage. Eukaryotic cells respond to replication interference through a complex signal-transduction pathway, known as the S-phase checkpoint, whose key players in S. cerevisiae are the Mec1 and Rad53 kinases. Both Mec1 and Rad53 are essential for budding yeast cell viability and mec1 and rad53 checkpoint mutants are extremely sensitive to agents that cause replicative stress, such as hydroxyurea (HU) and methyl methanesulfonate (MMS). The sensor kinase Mec1 is recruited to stalled replication forks, where it activates the effector kinase Rad53. The activation of both these kinases maintain the integrity/activity of the replication forks, stimulate deoxyribonucleotides (dNTPs) production, inhibit the firing of late replication origins and prevent accumulation of aberrant DNA structures. A fundamental question to be addressed was which of the above checkpoint-regulated process(es) is/are critical for the maintenance of cell viability. We investigated this question searching for extragenic mutations suppressing the hypersensitivity to HU of mec1 mutant. By characterizing one of the identified suppressor mutations, we provide evidence that decreased activity of Cdk1 alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G1 and mitosis. In fact, delaying either the G1/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to HU and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions. In conclusion, our results suggest that Cdk1 influences the DDR through multiple mechanisms. Indeed, Cdk1 is required for DSB-induced checkpoint activation, DSB repair by homologous recombination, and crossover formation. On the other hand, Cdk1 activity must be carefully regulated, because too much Cdk1 activity can affect genome integrity, at least when the checkpoint is not functional.