DNA folding by histones: the kinetics of chromatin core particle reassembly and the interaction of nucleosomes with histones.

Abstract The kinetics of the chromatin core particle reassembly reaction in solution were quantitatively studied under conditions such that nucleohistone aggregation did not occur. Core particles, salt-jumped rapidly by dilution from 2.5 m -NaCl (in which DNA and histones do not interact) to 0.6 m -NaCl (in which core particles are nearly intact), reassemble in two distinct time ranges. Approximately 75% of the DNA refolds into core particle-like structures “instantaneously” as measured by several physical and chemical techniques with dead times in the seconds to minutes time range. The remaining DNA refolds with relaxation times ranging from 250 minutes at 0 °C to 80 minutes at 37 °C; this slow effect cannot be attributed to sample heterogeneity. The fraction of slowly refolding DNA and the slow relaxation time are independent of the core particle concentration. Transient intermediates present during the slow phase of refolding were identified as free DNA and core particle-like structures containing excess histone. Mixing experiments with DNA, histones, and core particles showed that core particle-histone interactions are responsible for the slow kinetics of DNA refolding. Upon treatment of reassembling core particles with the protein crosslinking reagent, dimethylsuberimidate, the slow phase of the reassembly reaction was arrested and a 13 S particle containing DNA and two octamers of histone was isolated. Consistent with the nature of this kinetic intermediate, it is shown that in 0.6 m -NaCl, core particles co-operatively bind at least one additional equivalent of histones with high affinity in the form of excess octamers. Also, core particles continue to adsorb considerably more histones with a weaker association constant of the order 105 m −1 (in units of octamers) to a maximum value of 12 ± 2 equivalents (octamers) per core particle. The sedimentation coefficient increases with the two-thirds power of the molecular weight of the complex, as it would in the case of clustered spheres. A reassembly mechanism consistent with the data is presented, and other simple mechanisms are excluded. In the proposed mechanism, core particles reassemble very rapidly and compete effectively with DNA for histones such that approximately one-third of the particles initially formed are complexed with an excess octamer of histones, and 25% of the total DNA remains uncomplexed. The amount of this unusual reaction intermediate decays slowly to an equilibrium value of about 10%, thereby leaving 9% of the total DNA uncomplexed. Approximate values are calculated for the free energies, rate constants, and two of the activation energies which characterize this migrating octamer mechanism. This mechanism provides a means whereby histone octamers can be temporarily stripped off DNA at a modest free energy cost, approximately 2.6 kcal per nucleosome. Also, the properties of excess histone adsorption by chromatin and octamer migration suggest an efficient mechanism, consistent with observations by others, for nucleosome assembly in vivo during replication.