Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro.
Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro.
Smith
S
S
Cold Spring Harbor Laboratory, New York 11724.
Stillman
B
B
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002467:Cell Nucleus / Q000737:chemistry; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009687:Nuclear Proteins / Q000276:immunology*; D010750:Phosphoproteins / Q000737:chemistry; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D010750:Phosphoproteins