Cyclin B-Cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phas e support the semiconservative replication of supercoiled plasmids in vitro . We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initia tion in vivo. We found that replication in nuclear extracts initiates indep endently of the origin recognition complex (ORC), Cdc6p, and an autonomousl y replicating sequence (ARS) consensus. Nonetheless, quantitative density g radient analysis showed that S- and M-phase nuclear extracts consistently p romote semiconservative DNA replication more efficiently than G(1)-phase ex tracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extract s deficient for the Cdc7p kinase, In a cdc4-1 G(1)-phase extract, which acc umulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5 -Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G(1)-phase extract, In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), rest ores efficient replication to both extracts. Our results suggest that in ad dition to its well-characterized role in regulating the origin-specific pre replication complex, the Clb-Cdk1 complex modulates the efficiency of the r eplication machinery itself.