Comparison of replicative and non-replicative chromatin assembly pathways in HeLa cell extracts.
It has been reported that chromatin assembly in mammalian cell extracts depends exclusively or preferentially on ongoing DNA replication (Stillman, B. (1986) Cell 45, 555-565). More recently, this view has been challenged demonstrating that, in the same extracts, chromatin can also be formed efficiently in the absence of DNA replication (Gruss et al. (1990) EMBO J. 9, 2911-2922). The experiments, described in this communication, were performed to resolve this apparent contradiction. We found that there are at least two distinct in vitro pathways for chromatin assembly in HeLa cell extracts. The replicative pathway requires a nuclear protein, most likely identical with the chromatin assembly factor, described by Stillman (1986, Cell 45, 555-565), and the free soluble histones present in the cytosol of S phase cells. In contrast, a non-replicative pathway was identified that depends on isolated nuclear histones. As one component of the non-replicative assembly pathway we identified a cytosolic factor that was purified to apparent homogeneity and shown to be an acidic 50 kDa polypeptide. The isolated cytosolic 50 kDa protein efficiently promoted nucleosome assembly as demonstrated by one- and two-dimensional gel electrophoresis of in vitro packaged plasmid DNA.
Lässle
M
M
Division of Biology, Universität Konstanz, Germany.
Richter
A
A
Knippers
R
R
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology; D002474:Cell-Free System; D002843:Chromatin / Q000502:physiology* / Q000648:ultrastructure; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000378:metabolism; D004261:DNA Replication; D004278:DNA, Superhelical / Q000378:metabolism; D017886:Durapatite; D015180:Electrophoresis, Gel, Two-Dimensional; D006367:HeLa Cells; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D002843:Chromatin; D004278:DNA, Superhelical; D006657:Histones; D006882:Hydroxyapatites; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D017886:Durapatite