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 cerevisiae. 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.
Mimura
Satoru
S
Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3LD, UK.
Seki
Takashi
T
Tanaka
Seiji
S
Diffley
John F X
JF
D020816:Amino Acid Motifs; D001665:Binding Sites; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D019926:Cyclin B / Q000737:chemistry / Q000378:metabolism; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
C066503:CDC6 protein, S cerevisiae; C093749:CLB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D019926:Cyclin B; D016213:Cyclins; D010446:Peptide Fragments; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins; D017489:CDC28 Protein Kinase, S cerevisiae