Polo-like kinases (PLKs) have an important role in several stages of mitosis. They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis. PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis. Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint. Here we report the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines. We show that Plk1 is inhibited by DNA damage in G2 and in mitosis. In line with this, we show that DNA damage blocks mitotic exit. DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1. Significantly, expression of these mutants can override the G2 arrest induced by DNA damage. On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.

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DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

Chromatin association and dissociation appears to be important for proper Chk1 regulation. We propose that in response to DNA damage, PIKK-dependent checkpoint signaling leads to phosphorylation of chromatin-bound Chk1, resulting in its rapid release from chromatin and facilitating the transmission of DNA-damage signals to downstream targets, thereby promoting efficient cell-cycle arrest.