THE POLO-LIKE KINASE CDC5 AND THE CDK-COUNTERACTING PHOSPHATASE CDC14 PLAY DISTINCT ROLES IN THE RESOLUTION OF DNA LINKAGES IN MITOSIS

During mitosis, the newly duplicated genetic material, organized in pairs of sister chromatids, is distributed between the daughter cells by the spindle machinery, in a process called chromosome segregation. Errors in this process can compromise genome integrity. Faithful chromosome segregation requires the removal of all sorts of cohesion between sister chromatids. Although the main contributors of sister chromatid cohesion are cohesin complexes, which are cleaved at anaphase onset, another source of cohesion is represented by DNA linkages, also called Sister Chromatid Intertwines (SCIs). These linkages comprise unreplicated segments, recombination intermediates, and double-stranded catenanes. If not properly removed, SCIs can break during cell division causing DNA damage and jeopardizing genome stability. Although most DNA linkages are removed before mitosis, their complete resolution only occurs concomitantly with chromosome segregation, in a process whose regulation is still poorly understood. In this thesis, to investigate the mechanisms of SCI resolution during mitosis, we exploited the unique phenotype of S. cerevisiae cells lacking the activities of the polo-like kinase Cdc5 and the Cdk-counteracting phosphatase Cdc14. These cells arrest after cohesin cleavage, with short bipolar spindles and undivided nuclei, because impaired in spindle elongation. Evidence suggests that cdc5 cdc14 cells are also impaired in sister chromatid separation, due to the presence of unresolved SCIs, and previous work in our laboratory revealed that these linkages mainly consist of DNA catenanes. Here, we found that both Cdc14 and Cdc5 contribute to the resolution of DNA linkages, with different functions. Cdc14 is mainly involved in nucleolar segregation and processing of recombination intermediates, while Cdc5 seems to act through a more generalized mechanism and promote the removal of DNA catenanes. At the molecular level, Cdc14 acts through its known substrate Yen1. On the other hand, we found that Cdc5 controls post-translational modification of the decatenating enzyme Top2 during mitosis, particularly conjugation with small ubiquitin-like modifier (SUMO) and, possibly, also phosphorylation. The polo-like kinase is known to inactivate the SUMO protease Ulp2 in metaphase, thus increasing SUMOylation of Ulp2 substrates, like Top2. Since the decatenation defect of cdc5 cells correlates with a dysregulation of the SUMO pathway and this pathway is known to regulate sister chromatid cohesion, we speculate that the hyperactivation of Ulp2 may be the reason behind the sister chromatid separation defect of cdc5 cells. Taken together, our findings integrate the current knowledge of the mechanisms of sister chromatid separation and allow us to propose a model that foresees Cdc5 and Cdc14 coordinating cohesin cleavage and spindle elongation with the removal of DNA intertwines.