EARLY EVENTS IN DNA-REPLICATION REQUIRE CYCLIN-E AND ARE BLOCKED BY P21(CIP1)

Using immunodepletion of cyclin E and the inhibitor protein p21(WAF/CIP1), we demonstrate that the cyclin E protein, in association with Cdk2, is required for chromosomal replication in Xenopus extracts. The requirement for cyclin E occurs early in the replication process and cyclin E/Cdk2 is not required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.