Native chromatin-DNA structure and cell cycle: differential scanning calorimetry and gel electrophoresis

Abstract

Differential scanning calorimetry (DSC) and gel electrophoresis have been carried out on intact nuclei and on the corresponding chromatin extracted, using the two most common procedures, from rat hepatocytes, as a function of shearing, ionic strength and DNA-ase digestion. These studies, along with those obtained on free DNA fragments of widely different length, suggest that the chromatin prepared by limited nuclease digestion lacks the topological constraints present in situ, which appear instead to be preserved even at low ionic strength (0.01 M) by lysis of the native nuclei in cold hypotonic buffer and by subsequent gentle resuspension (without shearing) of the viscous chromatin mass. While both unsheared chromatin preparations contain a similar repeating structure, only the chromatin prepared by limited nuclease digestion is lacking the higher molecular-weight bands present in native nuclei. Furthermore only “cold water” chromatin reveals significant cell-cycle-related thermodynamic alterations which exactly mimic those apparent in situ from the heat capacity profiles of corresponding nuclei. Results are discussed in terms of DNA packing/supercoil in vivo which could modulate during cell-cycle progression and which could be maintained by the topological constraints existing along native nucleofilaments.