Influence of positive stress on nucleosome assembly

Nucleosome assembly on closed circular DNA has been studied using reconstitution protocols which involve salt gradient dialysis in the presence or absence of urea. The closed circular DNA contained either +3 or -14 supercoils. The nucleosomal content of the DNA was assayed by measuring the superhelical content in the presence of topoisomerase I as a function of time and temperature and by measuring the DNA fragment size after DNase I digestion. Positively coiled DNA appears to contain an altered "open" nucleosome structure as compared to the negatively coiled DNA. Formaldehyde fixation of the reconstituted complexes was also done in an attempt to freeze the nucleosomal state prior to topoisomerase I or DNase I addition. The data from these additional experiments are consistent with a conclusion that an altered nucleosomal structure exists on positively supercoiled DNA. Control experiments are included to verify the efficiency of formaldehyde fixation in preserving the structure in the reconstituted complexes. The physiological relevance of positive stress and its effect on nucleosome structure are discussed.