PREFERENTIAL BINDING OF HISTONES H3 AND H4 TO HIGHLY POSITIVELY COILED DNA

The interaction of histones H3 and H4 with highly positively coiled DNA has been studied. To carry out this study, it was necessary to develop a protocol for the preparation of large quantities of highly positively coiled DNA. Such preparations were obtained by maximizing transcription-induced stress in cells containing PBR322 and then applying a short-term treatment with novobiocin, which inhibits gyrase. Fractionation on CsCl-EtBr gradients gave PBR322 plasmids with topological states ranging from +0.15 to -0.043 superhelical density. In competition experiments between negatively and positively coiled DNA, histones H3,H4 preferentially bound the positively coiled DNA when the superhelical density was greater than +0.10. This preference was shown on the basis of sedimentation rate on sucrose gradients, selective aggregation by H3,H4 binding, and cross-linking experiments in which the histone-DNA content was characterized on CsCl density gradients. An analysis of the DNA helical pitch in 1.1 M NaCl, a condition in which moderately positively coiled DNA preferentially binds H3,H4, indicated that the preferential binding may be due to a decrease in helical pitch that approximates 0.06 bp/tum. Consistent with this observation is the ability of histones H3,H4 to transiently hold this altered pitch in the presence of topoisomerase I. A possible explanation for this preference is based on the known observation that histones overwind the DNA helix when in a nucleosome. These data provide an explanation for the in vivo observation that histones H3,H4 are rarely displaced during the transcription process. These observations are discussed with regard to mechanisms for transcription through nucleosomes.