BOTH CYCLIN-A AND CYCLIN-E HAVE S-PHASE PROMOTING (SPF) ACTIVITY IN XENOPUS EGG EXTRACTS

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affi nity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in the se Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A pr omoted entry into mitosis, which inhibited DNA replication. In contras t, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cycl in E1 complexes exclusively with Cdk2 and cyclin A is complexed predom inantly with Cdc2; only later in development does cyclin A associate w ith Cdk2. We show that baculovirus-produced complexes of cyclin A-Cdc2 , cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. The se results suggest that although in the early Xenopus embryo cyclin E1 -Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provi des a significant additional quantity of SPF as its levels rise during S phase.