Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase alpha.
Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
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Desdouets
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Santocanale
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Drury
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Perkins
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Foiani
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Plevani
J F
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Diffley
D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000502:physiology*; D002843:Chromatin / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D004256:DNA Polymerase I / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000502:physiology; D016193:G1 Phase / Q000502:physiology; D008938:Mitosis / Q000502:physiology*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017489:CDC28 Protein Kinase, S cerevisiae; D019915:DNA Primase; D004256:DNA Polymerase I