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Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control


Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae1,2. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.

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Figure 1: Clb2/Cdc28 inhibition of Cdc6 loading.
Figure 2: CDK consensus sites in the NTD prevent re-replication in vivo.
Figure 3: Phosphorylation-dependent binding of CDKs to Cdc6 N-terminal peptides.
Figure 4: Recombinant Clb2 binds specifically to Cdc6 phosphopeptides.


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We are grateful to members of our laboratory for discussions and for critical reading of the manuscript. We are also grateful to J. Gannon and T. Hunt for discussions as well as human CDK antibodies and p13suc1 beads. We thank L. Drury, K. Labib, J. Li, G. Perkins and S. Reed for yeast strains. We also thank N. O'Reilly and the Peptide Synthesis Facility at the London Research Institute. This work was supported by Cancer Research UK and the Human Frontier Science Program Organization. S.M. is supported by JSPS postdoctoral Fellowships for Research Abroad.

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Correspondence to John F. X. Diffley.

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Mimura, S., Seki, T., Tanaka, S. et al. Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control. Nature 431, 1118–1123 (2004).

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