Purification and mechanism of action of a nucleosome assembly factor from Xenopus oocytes.
Chromatin with nucleosomes spaced at 180-base pair intervals can be formed in vitro on circular DNA molecules using a Xenopus oocyte S-150 extract, but the ability to form a periodic chromatin structure is lost upon fractionation of this extract. To identify factors other than the known ones involved in chromatin assembly, we have first depleted the extract by incubating it in batch with charged resins, and we have subsequently reconstituted it with purified fractions. Studies performed with the fractionated components indicate that formation of periodically spaced nucleosomes on the relaxed, closed circular DNA proceeds in two steps and does not require DNA topoisomerases. In a first step, histones H3/H4 are transferred from the endogenous H3/H4-N1 complex to the DNA, forming a nascent chromatin structure. This structure can then be rapidly complemented in a subsequent and independent step with a stoichiometric amount of histone H2A/H2B dimers. Under these experimental conditions, excess histone H2A/H2B dimers inhibit DNA supercoiling and nucleosome formation. We describe the purification of a factor from the Xenopus oocyte S-150 which permits DNA supercoiling and nucleosome formation under conditions of excess histone H2A/H2B. The activity purifies as a complex of five nonacidic polypeptides with apparent molecular masses ranging between 56 and 62 kDa. This factor prevents the binding of excess histone H2A/H2B to the DNA, and it can also remove excess histone H2A/H2B already bound to the DNA, thus ensuring that stoichiometric amounts of all four nucleosomal histones associate with the DNA.
Sapp
M
M
Department of Biology, University of Rochester, New York 14627.
Worcel
A
A
D000818:Animals; D002411:Cation Exchange Resins / Q000494:pharmacology; D002458:Cell Fractionation; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure; D002875:Chromosomes / Q000648:ultrastructure; D004264:DNA Topoisomerases, Type I / Q000378:metabolism / Q000494:pharmacology; D004278:DNA, Superhelical / Q000378:metabolism; D005260:Female; D006657:Histones / Q000378:metabolism / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009707:Nucleosomes / Q000378:metabolism / Q000648:ultrastructure; D009865:Oocytes / Q000032:analysis* / Q000648:ultrastructure; D010455:Peptides / Q000302:isolation & purification* / Q000494:pharmacology / Q000502:physiology; D012117:Resins, Synthetic; D014982:Xenopus laevis
D002411:Cation Exchange Resins; D002843:Chromatin; D004278:DNA, Superhelical; D006657:Histones; D046911:Macromolecular Substances; D009707:Nucleosomes; D010455:Peptides; D012117:Resins, Synthetic; C026257:Bio-Rex 70; D004264:DNA Topoisomerases, Type I