Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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CDC2-CDC28 Kinases*
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Cell Cycle
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Chromatin Assembly Factor-1
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Chromosomal Proteins, Non-Histone*
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Cyclin A / physiology*
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Cyclin B / physiology
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Cyclin B1
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Cyclin D
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Cyclin E / physiology
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinases / physiology*
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Cyclins / physiology
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DNA / biosynthesis*
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DNA / drug effects
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DNA, Complementary / metabolism
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DNA-Binding Proteins / metabolism*
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Dose-Response Relationship, Drug
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Enzyme Inhibitors / pharmacology
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Glutathione Transferase / metabolism
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HeLa Cells
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Humans
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Kinetics
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Mitosis
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Models, Biological
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Nucleosomes / metabolism
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Okadaic Acid / pharmacology
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Phosphoprotein Phosphatases / metabolism*
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Phosphorylation
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Protein Phosphatase 1
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Protein Serine-Threonine Kinases / metabolism
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Protein Serine-Threonine Kinases / physiology*
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Proto-Oncogene Proteins*
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Purines / pharmacology
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / metabolism
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Roscovitine
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Vanadates / pharmacology
Substances
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CCNB1 protein, human
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CHAF1B protein, human
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Chromatin Assembly Factor-1
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Chromosomal Proteins, Non-Histone
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Cyclin A
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Cyclin B
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Cyclin B1
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Cyclin D
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Cyclin E
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Cyclins
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DNA, Complementary
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DNA-Binding Proteins
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Enzyme Inhibitors
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Nucleosomes
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Proto-Oncogene Proteins
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Purines
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Recombinant Fusion Proteins
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Recombinant Proteins
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Roscovitine
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Okadaic Acid
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Vanadates
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DNA
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Glutathione Transferase
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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CDK2 protein, human
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CDK4 protein, human
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase 4
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Cyclin-Dependent Kinases
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Phosphoprotein Phosphatases
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Protein Phosphatase 1