Role for cyclin A-dependent kinase in DNA replication in human S phase cell extracts

Cell cycle progression is regulated by cyclin dependent kinases, Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication, Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase, Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither, We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity, By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication, Moreover, specific p21(Waf1) mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts, Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation.