Minor groove binding ligands alter the rotational positioning of DNA fragments on nucleosome core particles

Abstract

We have used hydroxyl radical and DNase I footprinting to examine the interaction of four AT-selective minor groove binding ligands (Hoechst 33258, distamycin, netropsin and berenil) with DNA fragments which have been reconstituted with nucleosome core particles. Hydroxyl radical footprints of reconstituted tyrT DNA show that all four ligands induce changes in the phased cleavage pattern, consistent with the suggestion that they cause the DNA to rotate by 180 degrees on the nucleosome surface. This observation was confirmed by a series of hydroxyl radical and DNase I footprinting experiments on a synthetic DNA fragment containing five different (A/T)4 sites spaced ten bases apart, in phase with the nucleosomal repeat. This fragment produces a phased cleavage pattern when bound to the nucleosome cores, with minima in the AT regions, suggesting that these regions are positioned with their narrow minor grooves facing towards the protein surface. In the presence of the minor groove binding ligands the hydroxyl radical cleavage maxima are shifted by about five base-pairs. It appears that the ligands have caused the DNA to rotate by about 180 degrees on the protein surface; those DNA regions which were facing out are turned in and vice versa. Regions to which the ligands are bound are turned away from the protein surface, thereby minimising electrostatic repulsion between the cationic charges on the ligand and protein. The absence of any observable footprints in the AT-regions suggests that these changes are induced at low levels of occupancy.