Binding of actinomycin D to calf thymus chromatin

Abstract

Studies are made on the binding of actinomycin D to calf thymus DNA and chromatin. The effect upon binding of the removal of chromosomal protein by NaCl is examined. Protein is extracted from chromatin with 0.6, 1.0 and 2.0 m-NaCl, and is separated from the remaining nucleoprotein on a Bio-gel A50 column. The amount of total protein, histone and acid-insoluble protein removed at different salt concentrations is examined, and the type of histone removed is determined using analytical disc electrophoresis. Ultraviolet absorption spectra and melting profiles of the nucleoprotein samples are also taken. Actinomycin D binding to chromatin, salt-extracted chromatin, and DNA is examined spectrophotometrically, and the resulting data are used to graph Scatchard plots, from which both binding constants and the number of actinomycin D molecules bound to DNA are determined. Chromatin, like DNA, appears to have more than one set of binding sites, and the set of strongest binding sites appears to be identical for DNA and chromatin. DNA has close to three times as many strong binding sites as chromatin. The number of actinomycin D molecules bound to these strong binding sites shows a non-linear relationship with the percentage of total protein and histone protein removed, rising more rapidly per unit amount of protein when the last portions of protein are removed. The removal with salt of chromosomal protein not extractable with 0.4 n-H 2SO 4, which contains some histone as well as non-histone protein, shows a more linear relationship with the increase in actinomycin D binding.