BOTH CDC2 AND CDK2 PROMOTE S-PHASE INITIATION IN XENOPUS EGG EXTRACTS

Xenopus egg extracts induce S phase DNA replication in added sperm pro nuclei in a highly regulated manner, similar to events in vivo. Remova l of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have us ed p13(suc1) beads to remove both cdk2 and cdc2 proteins from egg extr acts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their r espective mRNAs, restimulated replication, judged by both total synthe sis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R3 3) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued rep lication in this system. Human mutant mRNAs have been used to show tha t the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34(cdc2) is al so observed in extracts depleted of cdks with a cdk2 antibody, which s till retain much of their endogenous cdc2 protein. We conclude that ne wly synthesised p34(cdc2), but not the inherited 'old' form, can induc e S phase and in this form may overlap in function with p33(cdk2).