NUCLEOSOME ASSEMBLY ACTIVITY AND INTRACELLULAR-LOCALIZATION OF HUMAN CAF-1 CHANGES DURING THE CELL-DIVISION CYCLE

We characterized changes of nucleosome assembly activity, intracellula r localization, and reversible phosphorylation of the human chromatin assembly factor CAF-1 during the somatic cell division cycle. HeLa cel ls were synchronized in the G(1), S, G(2), and M phases of the cell cy cle. All three subunits of human CAF-1 (p150, p60, and p48) are presen t during the entire cell cycle. In interphase, p150 and p60 are bound to the nucleus, but they predominantly dissociate from chromatin durin g mitosis, During S phase, p150 and p60 are concentrated at sites of i ntranuclear DNA replication. Only a fraction of total p48 is associate d with p150 and p60, and the majority is present in other high molecul ar weight complexes. The other nucleosome assembly protein, NAP-1, is predominantly cytosolic throughout the cell cycle, Human CAF-1 efficie ntly mediates nucleosome assembly during complementary DNA strand synt hesis in G(1), S, and G(2) phase cytosolic extracts. Active CAF-1 can be isolated as a 6.5 S complex from G(1), S, and G(2) phase nuclei. In contrast, CAF-1 isolated from mitotic cytosol does not support nucleo some assembly during DNA synthesis. In mitosis, the p60 subunit of ina ctive CAF-1 is hyperphosphorylated, whereas active CAF-1 in interphase contains hypophosphorylated and/or phosphorylated forms of p60.