Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract.
We report here a mammalian cell-free system that can support chromatin assembly. Effective nucleosome assembly in HeLa cell extracts occurred at 125 to 200 mM KCl or potassium glutamate. At this physiological K+ ion concentration, two types of chromatin assembly were observed. The first was interfered with by Mg2+. Other cations such as Mn2+, Ca2+, Fe3+, and spermidine also inhibited this type of nucleosome assembly. The second type of assembly occurred in the presence of Mg2+ and at least equimolar ATP. However, even in the presence of ATP, excess Mg2+ inhibited assembly and promoted catenation of DNA; these effects could be circumvented by excess ATP, GTP, EDTA, or polyglutamic acid. The critical DNA concentration for optimum assembly in both pathways suggested a stoichiometric association of histones with DNA. The spacing of nucleosomes formed by both types of assembly on linear and circular DNA was reasonably regular, but chromatin assembled in the presence of ATP and Mg2+ was more stable.
Banerjee
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Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000838:Anions; D002412:Cations; D002472:Cell Transformation, Viral; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism; D004278:DNA, Superhelical / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D004492:Edetic Acid / Q000494:pharmacology; D006367:HeLa Cells / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009707:Nucleosomes / Q000187:drug effects / Q000378:metabolism*; D009994:Osmolar Concentration; D013539:Simian virus 40 / Q000235:genetics* / Q000378:metabolism
D000838:Anions; D002412:Cations; D002843:Chromatin; D004278:DNA, Superhelical; D004279:DNA, Viral; D006657:Histones; D009707:Nucleosomes; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D008274:Magnesium