The effect of negative supercoiling on the formation and positioning of nucleosome cores in vitro

The effect of the negative supercoiling of DNA on the formation and positioning of nucleosome cores was investigated in a 1915bp plasmid (pHP2) containing a section of the early H1-H4 histone gene spacer of Psammechinus miliaris, previously shown to position the histone octamer (Retief et al., 1987, Biochemistry, 26, 4449-4453). It is shown for the first time, by determinations of the linking difference of reconstituted supercoiled plasmid following topoisomerase I relaxation and the yield and fragment size distribution of micrococcal nuclease digests of supercoiled and linearized plasmid, that nucleosome core reconstitution by urea/salt and salt dialysis proceeds cooperatively on both linearized and supercoiled plasmids. Evidence is further presented which indicates that the nucleosome core.reconstitutes more efficiently on negatively supercoiled plasmids compared to linearized plasmids. The free energy of supercoiling is shown to be sufficient to account for this difference, and may contribut·e to the observed preferential migration of the octamer to negatively supercoiled plasmid compared to linear fragments. This migration is facilitated by high ionic strength, but not by high concentrations of poly[L-glutamate] or 146bp core DNA. It is further shown for the first time, by DNase I digestion and primer extension, that identical translational and rotational positions are adopted by nucleosome cores on linearized plasmids and circular plasmids in the absence and presence of negative superhelical stress. This conservation of the positioning frames is -shown to persist, irrespective of the precision of the core placement, or alterations of the relative angular orientation of the positioning frames of adjacent cores. This finding suggests that the topological and geometric constraints of chromatin loops may be negligible in the determination of nucleosome positioning in vivo. The positioning of the core incorporating a d(A-G)₁₆.d(C-T)₁₆ stretch, shown not to adopt a H-DNA conformation in the reconstituted supercoiled plasmid, is analyzed in terms of known rotational determinants, and possible translational determinants proposed. The biological significance of the determined position of the core is discussed, and lastly, the conclusions related to other studies.

Grantor dc:publisher.institution

Department of Molecular and Cell Biology

Year dc:date.issued

1991

Author dc:creator

• Patterton, Hugh-George

Advisor dc:contributor.advisor

• Von Holt, Claus

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/11427/22097

OAI identifier oai:identifier

oai:open.uct.ac.za:11427/22097