[show abstract][hide abstract] ABSTRACT: In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G1 is necessary for subsequent initiation of DNA replication triggered by activation in late G1 of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6. The absence of pre-RCs during G2 and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G1 depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G1. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G2 and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated. We show that Cdc6p associates with Clb/Cdk1 kinases from late G1 until late anaphase, which might be important for inhibiting pre-RC assembly during S, G2, and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.
Genes & Development 07/1996; 10(12):1516-31. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: When yeast cells reach a critical size, they initiate bud formation, spindle pole body duplication, and DNA replication almost simultaneously. All three events depend on activation of Cdc28 protein kinase by the G1 cyclins Cln1, -2, and -3. We show that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins. A sextuple clb1-6 mutant arrests as multibudded G1 cells that resemble cells lacking the Cdc34 ubiquitin-conjugating enzyme. cdc34 mutants cannot enter S phase because they fail to destroy p40SIC1, which is a potent inhibitor of Clb but not Cln forms of the Cdc28 kinase. In wild-type cells, p40SIC1 protein appears at the end of mitosis and disappears shortly before S phase. Proteolysis of a cyclin-specific inhibitor of Cdc28 is therefore an essential aspect of the G1 to S phase transition.