CIP1 BLOCKS THE INITIATION OF DNA-REPLICATION IN XENOPUS EXTRACTS BY INHIBITION OF CYCLIN-DEPENDENT KINASES

Background: Cip1 is a 21 kD protein that interacts with and inhibits cyclin-dependent kinases (cdks). Expression of Cip1 is induced by the tumour suppressor p53, and tumour cells have greatly reduced levels of Cip1. As cdks are required for normal progression through the cell cycle, their inhibition by Cip1 may mediate the ability of p53 to block cell proliferation. Cip1 has also been shown to inhibit the DNA polymerase delta auxiliary factor PCNA (proliferating cell nuclear antigen), which is required for replication-fork elongation, and this could be an alternative mechanism by which p53-induced Cip1 blocks cell proliferation. Results: We have investigated the effect of Cip1 protein on chromosomal DNA replication, using cell-free extracts of Xenopus eggs that initiate and complete chromosome replication under normal cell-cycle control. Cip1 protein strongly inhibited an early stage of DNA replication in this system, and this inhibition was not complemented by extracts that had been affinity-depleted of cdks. In contrast, Cip1 did not inhibit the elongation of replication forks that had accumulated in the presence of aphidicolin. Cip1 inhibition of DNA replication was fully rescued by addition of cyclins A or E, but not cyclin B, cdk2 or PCNA. Conclusions: Our results suggest that Cip1 specifically blocks the initiation of DNA replication by inhibition of a cyclin-dependent kinase (cdk2), but has no major effect on the elongation of preassembled replication forks. The ability of cyclin A or cyclin E to rescue the Cip1 inhibition suggests that these cyclins may play a direct role in the initiation of replication in the Xenopus system.