Quantitative footprinting analysis of drug-DNA interactions: Fe(III) methidium-propyl-EDTA as a probe

Quantitative footprinting studies involving a 139-base pair restriction fragment from pBR322 DNA, a lexitropsin ligand and two different DNA cleavage agents, the enzyme DNase I and the footprinting reagent Fe(III) methidium-propyl-EDTA (Fe-MPE), are described. The autoradiographic data showed that the ligand, an analogue of netropsin possessing two N-methylimidazole groups, binds to four regions on the 139-mer which are rich in GC. Analysis of the data leading to individual binding constants for each of the four loading events on the 139-mer revealed that Fe-MPE and DNase I report the same binding constants for the lexitropsin bound to its interaction sequences. The fact that the data from both probes can be analyzed using a common model indicates that the DNA cleavage specificity of the probe and not its binding/cleavage mechanism is the important factor in reporting of site loading information in the footprinting experiment. The study also showed that under certain conditions it is possible to gain information on the density of ligand binding sites on carrier DNA by monitoring site loading events on the labeled fragment.