Linkage reduction allows reconstitution of nucleosomes on DNA microdomains

We have established an experimental system for reconstitution of an individual nucleosome on a closed DNA microdomain (operationally defined as a DNA domain of a size so small as to be unable to establish titratable superhelical turns). The microdomain (185 base-pairs (bp), composed of 128 bp encompassing the central part of the Saccharomyces cerevisiae ADH II promoter plus 57 bp of a polylinker) was obtained by ligation under conditions that produced three circularized forms characterized by different linkage numbers. These linkomers were tested for nucleosome reconstitution with S. cerevisiae histones. It was observed that only microcircles with linkage reduction (delta Lk = 1 or 2) could form a nucleosome, as defined by protection of a 145(+/- 2) bp DNA fragment from micrococcal nuclease, relaxed forms (open or closed circles) could not.