Studies on rates of nucleosome formation with DNA under stress

Rates of nucleosome formation on DNA with various superhelical topologies were determined using an in vitro deposition system. The core histones, H3, H2B, H2A, and H4 were reconstituted with polyglutamic acid (a deposition vehicle) and added to closed-circular DNAs. Aliquots were taken during the incubation, added to excess topoisomerase I, and incubated for 20 s to determine the number of negative coils constrained into nucleosome structure by the histones. When negatively supercoiled DNA was used, nucleosomes were formed virtually instantaneously in a temperature-independent process with all the negative coils being trapped into nucleosome structure. When relaxed DNA (no negative coils) was used, nucleosome formation was severely retarded. If topoisomerase I was maintained in the total incubation of histones with the relaxed DNA, nucleosome formation occurred with a measurable rate in a temperature-dependent process (activation energy of 17 kcal/mol). Similar observations were made when the method of reconstitution of histones with DNA involved salt gradient dialysis. The data indicates that the lack of preformed negative coils is a rate-limiting step in nucleosome formation and that when positive coils are also maintained within the circular DNA, these positive coils prevent nucleosome formation. The effect of DNA topology on nucleosome formation is discussed with regard to its physiological relevance in replication and transcription processes.