Unfolding of nucleosomes by ethidium binding.
We report spectroscopic, hydrodynamic, and biochemical studies on the complex of ethidium bromide with 140 base pair nucleosomal core particles. Fluorescence titration indicates a greater intrinsic affinity of ethidium for nucleosomes than for DNA, and fluorescence depolarization measurements imply increased immobilization of ethidium bound to nucleosomes, but with more extensive dye-dye energy transfer compared to DNA-bound dye. Ethidium intercalated into DNA in nucleosomes has a limiting reduced linear dichroism of -0.45 at 320 nm and -0.25 at 530 nm. Both the energy transfer and dichroism results are consistent with clustering of the nucleosome-bound dye molecules. Electric dichroism measurements and ultracentrifugation studies reveal that structural distortion of the nucleosome accompanies ethidium binding, occurring in the range of r (ethidium residues per base pair) values from 0.02 to 0.06. The distortion transition is characterized by an increase in the negative limiting reduced dichroism from 0.29 to 0.45 at 265 nm, an increase in the field-induced viscosity-limited rotational orientation time from 0.8 to 3 mus, and a decrease in sedimentation coefficient from 10.5 to 8.2 S. The complex was modeled hydrodynamically as a cylinder of 335-A length and 67-A diameter, containing 1.4 superhelical turns of DNA. Dimethylsuberimidate cross-linked nucleosomes, or native nucleosomes in the presence of Mg2+, bind ethidium weakly and are not distorted. The periodicity of cutting sites produced by DNase II digestion of nucleosomes remains constant as ethidium is added, but the bandwidth increases. A thermodynamic model is proposed to interpret the binding isotherm, based on enhancement of drug binding affinity due to release of superhelical stress in the nucleosome-ethidium complex.
D001482:Base Composition; D004247:DNA / Q000032:analysis; D004996:Ethidium / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium; D008433:Mathematics; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000187:drug effects / Q000648:ultrastructure*; D013050:Spectrometry, Fluorescence
D009707:Nucleosomes; D004247:DNA; D004996:Ethidium; D008274:Magnesium