Studies of ColE1-plasmid DNA and its interactions with histones: sedimentation velocity studies of monodisperse complexes reconstituted with calf-thymus histones

Abstract

Complexes between the four calf-thymus histones (H2A, H2B, H3 and H4) and colE1-plasmid DNA have been reconstituted using the procedure of Oudet et al. ((1975), Cell 4, 281-300). The sedimentation rates of the complexes formed were studied under a variety of conditions. In 0.4 MNaCL, 0.1 M Tris pH 7.50, 0.01 M EDTA and 0.02 M NaHSO3, the final dialy-sis-solvent in the reconstitution procedure, the sedimentation coefficients s23 were found to increase when the complexes were reconstituted at increasing histone to DNA ratios. True plateau regions were reached in the case of the relaxed circular and linear forms of the plasmid DNA. The sedimenting boundaries observed for the complexes at saturation are sharp, reflecting a narrow distribution of sedimentation coefficients and a homogeneity of the complex comparable to that of the uncomplexed DNA. Studies of the dependence of s 23 on the concentration of the complex at constant DNA to histones ratio have been undertaken at salt concentrations between 0.4 and 1.5 M NaCL in the above solvent. The formation of the complexes is reversible, at least at the higher ionic strengths. At salt concentrations below 0.36 M the complex precipitates from solution. Omission of histone H4 from the reconstitution mixture abolishes complex formation.