Requirement of Cyclin/Cdk2 and protein phosphatase 1 activity for chromatin assembly factor 1-dependent chromatin assembly during DNA synthesis.
The influence of reversible protein phosphorylation on nucleosome assembly during DNA replication was analyzed in extracts from human cells. Inhibitor studies and add-back experiments indicated requirements of cyclin A/Cdk2, cyclin E/Cdk2, and protein phosphatase type 1 (PP1) activities for nucleosome assembly during DNA synthesis by chromatin assembly factor 1 (CAF-1). The p60 subunit of CAF-1 is a molecular target for reversible phosphorylation by cyclin/Cdk complexes and PP1 during nucleosome assembly and DNA synthesis in vitro. Purified p60 can be directly phosphorylated by purified cyclin A/Cdk2, cyclin E/Cdk2, and cyclin B1/Cdk1, but not by cyclin D/Cdk4 complexes in vitro. Cyclin B1/Cdk1 triggers hyperphosphorylation of p60 in the presence of additional cytosolic factors. CAF-1 containing hyperphosphorylated p60 prepared from mitotic cells is inactive in nucleosome assembly and becomes activated by dephosphorylation in vitro. These data provide functional evidence for a requirement of the cell cycle machinery for nucleosome assembly by CAF-1 during DNA replication.
Keller
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Wellcome/Cancer Research Campaign Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom.
Krude
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D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D019925:Cyclin A / Q000502:physiology*; D019926:Cyclin B / Q000502:physiology; D056744:Cyclin B1; D056741:Cyclin D; D019927:Cyclin E / Q000502:physiology; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D016213:Cyclins / Q000502:physiology; D004247:DNA / Q000096:biosynthesis* / Q000187:drug effects; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008938:Mitosis; D008954:Models, Biological; D009707:Nucleosomes / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D011687:Purines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D000077546:Roscovitine; D014638:Vanadates / Q000494:pharmacology
C534699:CCNB1 protein, human; C533593:CHAF1B protein, human; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D056741:Cyclin D; D019927:Cyclin E; D016213:Cyclins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009707:Nucleosomes; D011518:Proto-Oncogene Proteins; D011687:Purines; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000077546:Roscovitine; D019319:Okadaic Acid; D014638:Vanadates; D004247:DNA; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1