Energetics and affinity of the histone octamer for defined DNA sequences

Previous studies have compared the relative free energies for histone octam er binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into fre e DNA and historic octamers (or free histones) on dilution without generati on of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (< 400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome r econstitution studies, human alpha -satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits Kd(s) of 0.03 and 0.06 nM, resp ectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are similar to2 kcal/mol more stable than total NCPs isolated from cellular chr omatin. As for mixed sequence NCPs, increasing ionic strength or temperatur e promotes dissociation. van't Hoff plots of K(a)s versus temperature revea l that the difference in binding free energy for lambda -satellite and 5S N CPs compared to bulk NCPs is due almost entirely to a more favorable entrop ic component for NCPs formed on the unique sequences compared to mixed-sequ ence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of r ecombinant tailless histones.