Cdc5 kinase activity links cell cycle regulation with genome stability

In response to DNA damage, all eukaryotic organisms activate a surveillance mechanism, called DNA damage checkpoint (DDC), to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to the damaged sites, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Studies both in yeast and mammals have involved the Polo-like kinase 1 (PLK1; Cdc5 in yeast) in turning off the DNA damage checkpoint and promoting the cell cycle re-start after DNA damage mediated checkpoint arrest. To further characterize the Cdc5 pathway in preserving genome stability, we investigated the cdc5-T238A mutation, which abolishes the in trans-phosphorylation by an unknown kinase in the activation loop of the kinase domain. Firstly, we found that the Cdc5-T238A protein variant has a lower kinase activity by in vitro assay without affecting cell cycle dependent protein stability. Also, we found that the cdc5-T238A cells have a mild G2/M delay in unperturbed conditions. Of importance, cdc5-T238A cells show severe defects in DDC inactivation and cell cycle restart after one persistent DNA double strand break (DSB) and uncapped telomeres. Interestingly, this checkpoint adaptation defect is associated to a significant delay in localization of Cdc5-T238A protein variant at spindle pole bodies. Furthermore, genetic analysis indicates that cdc5-T238A cells have prolonged activation of multiple mitotic checkpoint factors, such as Bfa1, Mad2 and Cdh1, after one irreparable DSB. Finally, we found that cdc5-T238A cells have a significant increase in chromosome loss and gross chromosomal rearrangement rates, which are features of genome instability. In particular, genetic and biochemical analysis indicate that the cdc5-T238A cells have compromised activity of the Mus81-Mms4 pathway, which is involved in the resolution of recombination intermediates during stressful replication and in response to DNA damage. Further characterization of this and other cdc5 alleles in yeast promises to be informative to elucidate the functional role of PLK1 in preserving genome stability in mammals, and to develop novel cancer therapy approaches.