Physical properties of DNA and chromatin isolated from G1- and S-phase HeLa S-3 cells. Effects of histone H1 phosphorylation and stage-specific nonhistone chromosomal proteins on the molar ellipticity of native and reconstituted nucleoproteins during thermal denaturation

Abstract

To help delineate how changes in chromatin organization are related to DNA replication and transcription during the HeLa S-3 cell cycle, we have extended previous studies of the composition and structure of chromatin in synchronized G1- and S-HASE CELLS. By analyzing changes in molar ellipticity at 276 nm ([theta 276]) during thermal denaturation, it was found that double-helical DNA molecules in native chromatin have different optical activities and thermal stabilities at these two stages of the cell cycle. Furthermore, profiles of d[theta 276]/dT vs. T indicate that native G1- and S-phase chromatins contain different families of DNA superstructures. To help determine the causes and functional significance of these chromatin reorganizations during the cell cycle, we compared the optical activities and thermal stabilities of DNA in native chromatin with protein-free DNA and DNA in nucleoproteins reconstituted in vitro by NaCl-urea gradient dialysis. In addition, we examined levels of histone phosphorylation, histone acetylation, and types of histone and nonhistone chromosomal proteins (NHCP) found in G1- and S-phase cells and in purified hydroxylapatite, (HAP) fractions of these nuclear proteins which were used for in vitro reconstitution. The results of the present studies indicate that changes in H1-DNA-NHCP interactions occur in vivo, are associated with the phosphorylation of histone 1 molecules, and appear to be responsible for the relaxation of compact G1-phase chromatin superstructures into more open S-phase configurations during the HeLa S-3 cell cycle.