Requirement of Cyclin/Cdk2 and Protein Phosphatase 1 Activity for Chromatin Assembly Factor 1-dependent Chromatin Assembly during DNA Synthesis

• Published in: The Journal of biological chemistry 2000-11, Vol.275 (45), p.35512-35521
• Main Authors: Keller, Christian, Krude, Torsten
• Format: Article
• Language: English
• Subjects: CAF-1 protein; CDC2-CDC28 Kinases; Cdk2 protein; Cell Cycle; Chromatin Assembly Factor-1; Chromosomal Proteins, Non-Histone; Cyclin A - physiology; Cyclin B - physiology; Cyclin B1; Cyclin D; Cyclin E - physiology; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases - physiology; Cyclins - physiology; DNA - biosynthesis; DNA - drug effects; DNA, Complementary - metabolism; DNA-Binding Proteins - metabolism; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Glutathione Transferase - metabolism; HeLa Cells; Humans; Kinetics; Mitosis; Models, Biological; Nucleosomes - metabolism; Okadaic Acid - pharmacology; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Phosphatase 1; Protein-Serine-Threonine Kinases - metabolism; Protein-Serine-Threonine Kinases - physiology; Proto-Oncogene Proteins; Purines - pharmacology; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - metabolism; Roscovitine; Vanadates - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M003073200

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.