Negative supercoiling and nucleosome cores. II. The effect of negative supercoiling on the positioning of nucleosome cores in vitro.

The influence of unrestrained negative superhelical stress on nucleosome core positioning was investigated in vitro for a core located on a section of the early H1-H4 histone gene spacer of Psammechinus miliaris. We show that the position of this core on a reconstituted molecule occupied by 11 nucleosome cores is identical on a linear DNA molecule and a circular DNA molecule in the absence of unrestrained negative superhelical stress. This position is also identical to that previously found on a 337 base-pair fragment of corresponding sequence. We conclude that the core position is determined primarily by the DNA sequence, and is not influenced by core-core interactions or spatial constraints imposed by an altered geometry of the DNA molecule. This finding is supported by the identical positions assumed by the nucleosome core after altering the angular orientation of the DNA molecule, and presumably that of adjacent cores, on either one or both sides of the test core. It is further demonstrated that the core on the histone spacer region assumes identical positions on circular DNA molecules in both the presence and absence of excess negative superhelical stress equivalent to sigma = -0.03. This result indicates that conservative levels of negative supercoiling do not induce a shift in the positions of nucleosome cores. The biological implications of the experimental results are discussed and related to the findings of other workers.