Visualising the dissociation of sequence selective ligands from individual binding sites on DNA

Abstract

We have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time-dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37 ° C.Key words." Echinomycin; Mithramycin; Nogalamycin; Dissociation that the complexity observed with natural DNA is due to the parallel dissociation from different binding sites, each of which has different microscopic kinetic constants.We have recently developed a modification of the footprinting technique [20] for visualising the dissociation of ligands from individual binding sites in a mixed sequence DNA. In this technique a complex between the ligand and radiolabelled DNA is dissociated by addition of excess unlabelled DNA. Samples are removed from the reaction mixture at various times and subjected to DNase I footprinting. Dissociation from each binding site is visualised as the time-dependent disappearance of the footprint. In this paper we use this technique to examine the dissociation of echinomycin, mithramycin and nogalamycin from tyrT DNA, and obtain the first estimates for the dissociation of these ligands from individual binding sites. Materials and methods

Kinetic studies on the formation of DNA triplexes containing the nucleoside analogue 2′-O-(2-aminoethyl)-5-(3-amino-1-propynyl)uridine

“…Dissociation was initiated by adding a 10-fold excess of an oligonucleotide complementary to the third strand, thereby sequestering the free TFO as a short duplex. 24, 41 Since duplex formation is much faster than triplex formation, the rate of disappearance of the footprint corresponds to the dissociation of the third strand from its target. The reaction was followed by removing 3 ll aliquots at various time points (between 30 sec and 4 h) and digesting with DNase I as above.…”

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction

“…Similarly, Mith differentially interacted with individual G/C tetranucleotide sites, as revealed by DNase and hydroxyl radical probing [16][17][18].…”

“…However, potential binding sites for the antibiotic in the genomic DNA are not restricted to self-complementary sequences. Similarly, Mith differentially interacted with individual G/C tetranucleotide sites, as revealed by DNase and hydroxyl radical probing [ 16 , 17 , 18 ].…”