Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro

S Smith,B Stillman

doi:10.1016/s0021-9258(18)99062-9

Abstract

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.

Highlights

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 uitro
The monoclonal antibodies used as controls in the experiments were:pAb419, a monoclonal antibody directed against SV40 T antigen, previously called L19 (Harlow et al, 1981); mAbp70-9, a monoclonal antibody directed against a 70-kDa polypeptide in replication factor A (Fairman and Stillman, 1988; Din et al, 1990); and mAb 1644, a monoclonal antibody against human DNA polymerase CY,originally designated as SJK287-38 (Tanaka et al, 1982)
The polypeptide composition of purified CAF-I is shown in Fig. L4, lane 1

Summary

MATERIALS ANDMETHODS

Monoclonal Antibodies-A highly purified fraction of CAF-I, prepared as described previously (Mono Q , fraction 6 of the purification, Smith and Stillman (1989)),was used to immunize a BALB/c mouse. The mousewas immunized three times during a four week period with purified CAF-I antigen (5 pgof each CAF-I polypeptide per injection) in complete Freund’s adjuvant (primary injection) or incomplete Freund‘s adjuvant (subsequent injections)i,ntraperitoneally. This was followed by two injections (one intraperitoneal and the otherintravenous) of 5 pg each of purified CAF-I antigen in phosphate-buffered saline (PBS). The second and third screens were for those antibodies that recognized CAF-I polypeptides in an immunoblotting analysis (described below) using purified CAF-I antigen and/. ’ The abbreviations used are: CAF-I, chromatin assembly factor I; mAb, monoclonal antibody; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate

Chromatin Assemblyin Vitro

RESULTS

Nuclear Nuclear

IC d

DISCUSSION