Abstract
Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCFCDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.
Highlights
To constitute the pre-replicative complex (pre-RC) and initiate DNA replication, all sixcomponents of the Origin Recognition Complex (Orc1-6p) bind to replication origins followed by Cdc6p, Cdt1p and the Mcm2-7p complex [1]
We show a mechanism by which DNA re-replication is controlled by Cyclin Dependent Kinase (CDK) and a yeast glycogen synthase kinase-3 (GSK-3) kinase (Mck1p) in S. cerevisiae
Mck1p targets Cdc6p through a GSK-3 consensus site (T368), and Cdc6p protein degradation was mediated through the same T368 site
Summary
To constitute the pre-RC and initiate DNA replication, all sixcomponents of the Origin Recognition Complex (Orc1-6p) bind to replication origins followed by Cdc6p, Cdt1p and the Mcm2-7p complex [1]. The pre-RC has to be activated by the Dbf kinase-Cdc7p complex, resulting in the formation of a bidirectional replication fork in which the Mcm complex acts as a replicative helicase [1]. DNA polymerase synthesizes new strands of DNA. The cell cycle progression is driven by the Cyclin/CDK complex. Of the nine cyclins in S. cerevisiae six are Btype cyclins (Clb1-6) [2] and there is a single CDK (Cdc). Cdc28-Clb activity is required to initiate DNA replication [3,4,5]
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