Negative supercoiling and nucleosome cores. I. The effect of negative supercoiling on the efficiency of nucleosome core formation in vitro

Abstract

The efficiency of nucleosome core formation in vitro as a function of DNA topology was investigated. We show that the reconstitution of nucleosome cores by urea/salt dialysis on both negatively supercoiled and linearized plasmid proceed co-operatively, and that negatively supercoiled molecules are reconstituted significantly more efficiently compared with linearized molecules. The free energy of supercoiling, related to the square of the linking deficit, is further shown to be sufficient to account for this difference, which is particularly pronounced at low molar reconstitution ratios of octamer: DNA. At these low molar ratios the average number of cores formed per negatively supercoiled molecule is equal to the input ratio of octamer: DNA, in contrast to linearized molecules, where few if any cores are reconstituted under identical experimental conditions. The possible contribution of supercoil-stabilized non-B-DNA structural transitions to differences in core-DNA interactions on supercoiled and linearized DNA was also investigated. We show that the change in the nuclease susceptibility of a d(A-G).d(C-T) run in the free and reconstituted supercoiled plasmid is consistent with the reversion of the poly(purine).poly(pyrimidine) stretch from an H-DNA form to a B-DNA form following reconstitution of the negatively supercoiled plasmid into nucleosome cores. The biological significance of the supercoil-dependent efficiency of core formation is discussed, and the results related to other work.