Reassociation of histone H1 to H1-depleted polynucleosomes.

A new procedure is described for the reassociation of histone H1 to rat liver polynucleosomes selectively depleted of H1 and nonhistone proteins. The fidelity of reconstitution was scrutinized by nuclease digestion and sedimentation studies monitoring the regeneration of structural features which disappear following removal of H1. The results demonstrate that the 166-base pair barrier of digestion with micrococcal nuclease was restored in polynucleosomes reconstituted at the same ratio of H1 per octamer as that found in native nuclei, while association of twice that amount of H1 produced a barrier of digestion at about 178-180 base pairs. Polynucleosomes associated with polylysine exhibited a similar barrier as H1-depleted polynucleosomes. Digestion of H1-reconstituted polynucleosomes with DNase I produced preferentially dinucleosomal DNA fragments in the same manner as that of untreated polynucleosomes. The rate of digestion of H1-reconstituted polynucleosomes by micrococcal nuclease and DNase I was also comparable to that of untreated polynucleosomes. Sedimentation of H1-reconstituted polynucleosomes in a quasi-physiological ionic milieu revealed the regeneration of disassembly-refractory particles, a structural feature of untreated polynucleosomes. We conclude that the nucleosomal and supra-nucleosomal structure of polynucleosomes was reconstituted with fidelity.