Characterization of the CDC14 Phosphatase in the DNA damage response

Resumen

In our laboratory we are interested in deciphering the role of the protein phosphatase Cdc14 in the DNA damage response (DDR) in Saccharomyces cerevisiae. In summary, we have found that Cdc14 is essential for cell viability when cells are exposed to different genotoxic stresses, independently of its role in an unperturbed cell cycle. Importantly, the phosphatase is transiently released to the nucleoplasm in the presence of one/multiple double-strand breaks (DSBs). This change in the localization pattern of Cdc14 coincides with Net1 phosphorylation, a pre-requisite for the liberation of the phosphatase in a normal cell cycle by the mitotic exit network (MEN). Surprisingly, Cdc14 shuttling in the presence of DNA damage is limited to the nucleus and does not promote the mitotic exit. Additionally, Net1 remains phosphorylated even when Cdc14 signal is localized at the nucleolus again after its release in the presence of DNA damage. These findings suggest that additional mechanisms may govern Cdc14 re-localization during the DDR. Nevertheless, we did not explore them. There are two non-mutually exclusive explanations for the essential role of Cdc14 in the context of DNA damage: 1) an involvement in the DNA damage checkpoint activation; or 2) a direct role of the phosphatase in DNA repair. We first tested for the capability of cdc14-1 cells to activate the DNA damage checkpoint by measuring mononucleated G2/M arrested cells and Rad53 phosphorylation. We did not find differences between cells lacking the function of the phosphatase and the isogenic wild-type, indicating that Cdc14 is not required for a proficient DNA damage checkpoint activation. We next tested the efficiency of DNA repair by using different systems based on the generation of a repairable DSB by the endonuclease HO, previously described in the budding yeast. We found that Cdc14 activity is required to efficiently repair DSBs by homologous recombination (HR) both by using synthesis-dependent strand annealing (SDSA) and double strand break repair (DSBR). Importantly, Cdc14 is not associated with an increase/decrease in crossover formation and has no role in the DNA repair pathway choice. In order to find out what is the direct role of the phosphatase during the damage response we performed mass spectrometry with phospho-enrichment to look for proteins that were hyperphosphorylated in cdc14-1 mutants. We found proteins associated with the spindle-pole body (SPB), with the recognition of the break and the processing of the DNA molecule. It is important to remark that all the results mentioned above and other findings that are not included in this thesis were published in The EMBO journal [1]. In this study, we focused on the validation of the essential nuclease Dna2 as a Cdc14 target during the DDR. This nuclease, together with the helicase Sgs1 and other proteins, participates in the DNA end resection process that occurs at the beginning of the HR pathway. We found that Dna2 was hyperphosphorylated in cdc14-1 mutants thus corroborating our mass spectrometry data. Strikingly, while DNA resection was efficiently activated and inactivated in cells lacking Cdc14 activity, it remained active in cdc14-1 cells thus suggesting that the phosphatase might be involved in the inhibition of the DNA resection machinery (unpublished data). Importantly, deletion of Exo1, a redundant pathway that also participates in DNA resection, did not rescue the phenotype observed in cdc14-1 mutants. Finally, deletion of Dna2 by using the auxin-inducible degron (AID) rescued the hyper-resection phenotype of cells lacking Cdc14 activity (unpublished data). Altogether, we have found that Cdc14 is transiently released and activated during the DDR. The re-location of the phosphatase is necessary for the inhibition of the Dna2-Sgs1 pathway and, in consequence, for the proficient activation of the DNA repair pathway. In addition, we found that the phosphatase is required for the stabilization of the metaphase spindle to allow the recruitment of DSBs to the SPB periphery. Indeed, this DSB-SPB interaction is necessary to execute a proficient DNA repair by HR [1]. Even if we did not include these last findings in this work they could be related with the hyper-resection phenotype observed in cdc14-1 cells. In this line, it would be interesting to test whether if these two functions of Cdc14 are connected or not. 1. Villoria MT, Ramos F, Duenas E, Faull P, Cutillas PR, Clemente-Blanco A: Stabilization of the metaphase spindle by Cdc14 is required for recombinational DNA repair. EMBO J 2017, 36:79-101.