S-PHASE-PROMOTING CYCLIN-DEPENDENT KINASES PREVENT RE-REPLICATION BY INHIBITING THE TRANSITION OF REPLICATION ORIGINS TO A PRE-REPLICATIVE STATE

Background: DNA replication and mitosis are triggered by activation of kinase complexes, each made up of a cyclin and a cyclin-dependent kinase (Cdk). It had seemed possible that the association of Cdks with different classes of cyclins specifies whether S phase (replication) or M phase (mitosis) will occur. The recent finding that individual B-type cyclins (encoded by the genes CLB1-CLB6) can have functions in both processes in the budding yeast Saccharomyces cerevisiae casts doubt on this notion. Results: S, cerevisiae strains lacking Clb1-Clb4 undergo DNA replication once but fail to enter mitosis. We have isolated mutations in two genes, SIM1 and SIM2 (SIM2 is identical to SEC72), which allow such cells to undergo an extra round of DNA replication without mitosis. The Clb5 kinase, which promotes S phase, remains active during the G2-phase arrest of cells of the parental strain, but its activity declines rapidly in sill? mutants. Increased expression of the CLB5 ene prevents re-replication Thus, a cyclin B-kinase that promotes DNA replication in G1-phase cells can pr-event re-replication in G2-phase cells. Inactivation of Clb kinases by expression of the specific Clb-Cdk1 inhibitor p40(SIC1) is sufficient to induce a pre-replicative state at origins of replication in cells blocked in G2/M phase by nocodazole. Re-activation of Clb-Cdk1 kinases induces a second round of DNA replication. Conclusions: We propose that S-phase-promoting cyclin B-Cdk complexes prevent re-replication during S, G2 and M phases by inhibiting the transition of replication origins to a pre-replicative state. This model can explain both why origins 'fire' only once per S phase and why S phase is dependent on completion of the preceding M phase.