The Cdc25 A phosphatase is required for the G 1 -S transition of the cell cycle and is overexpressed in human cancers. We found that it is ubiquitylated and rapidly degraded by the proteasome and that its levels increase from G 1 until mitosis. By treating cells with the DNA synthesis inhibitor hydroxyurea, Cdc25 A rapidly decreased in abundance, and this was accompanied by an increase in Cdk2 phosphotyrosine content and a decrease in Cdk2 kinase activity. Cdc25 A overexpression altered the ability of cells to arrest in the presence of hydroxyurea, and caused them to undergo premature chromosome condensation. Cdc25 A overexpression could render tumor cells less sensitive to DNA replication checkpoints, thereby contributing to their genomic instability.

It is known that the direct binding of the cyclin-dependent kinase (Cdk) inhibitor p21, also called Cdk-interacting protein 1 (p21), to proliferating cell nuclear antigen (PCNA) results in the inhibition of PCNA-dependent DNA synthesis. We provide evidence that p21 first inhibits the replication factor C-catalyzed loading of PCNA onto DNA and second prevents the binding of DNA polymerase delta core to the PCNA clamp assembled on DNA. The second effect contributes most to the inhibition of pol delta holoenzyme activity. p21 primarily inhibited the DNA synthesis resulting from multiple reassembly of DNA polymerase delta holoenzyme. On the other hand, an ability of the PCNA clamp to translocate along double-stranded DNA was not affected by p21. These data were confirmed with a mutant of p21 that is unable to bind PCNA and therefore neither inhibited clamp assembly nor prevented the loading of DNA polymerase delta core onto DNA. Our data suggest that p21 does not discriminate in vitro "repair" and "replication" DNA synthesis based on template length but does act preferentially on polymerization which encounters obstacles to progress.

CDC25 dual-speci®city phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In human cells, CDC25A and C are involved in the control of G1/S and G2/M respectively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidence for an interaction between CDC25 phosphatases and members of the 14-3-3 protein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that phosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phosphatases and 14-3-3 proteins. We show that in the two-hybrid assay all three splice variants of CDC25B interact similarly and strongly with 14-3-3Z, b and z proteins, but poorly with e and y. In vitro, CDC25B interacts at a low level with 14-3-3b, e, z, Z, and y isoforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a speci®c, strong interaction between CDC25B and 14-3-3z and Z isoforms is revealed by a deletion of 288 residues in the amino-terminal region of CDC25B. This interaction requires the integrity of Ser 323, although it is independent of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is dierent from that with CDC25C. We propose that, in addition to a low anity binding site that is available for all 14-3-3 isoforms, post-translational modi®cation of CDC25B in vivo exposes a highanity binding site that is speci®c for the z and Z14-3-3 isoforms.