In footprinting experiments, an increase in DNA cleavage with addition of ligand to a system may be due to a ligand-induced structural change. Ligand binding also enhances cleavage by displacing the cleavage agent from ligand-binding sites, thus increasing its concentration elsewhere. The theory and characteristics of this mass-action enhancement are given, and it is shown how it may be recognized. Results of DNase I footprinting of small oligomers, with actinomycin D as ligand, are analyzed to reveal which enhancements are due to mass action, and which can reasonably be ascribed to structural changes. Patterns in the footprinting plots from our experiments on actinomycin D binding to a 139-base-pair DNA fragment (with DNase I as a probe) are studied in the same way. The likely origins of these patterns are discussed, as are enhancements occurring with other probes commonly used in footprinting experiments.
Goodisman, J., Rehfuss, R., Ward, B., & Dabrowiak, J. C. (1992). Site-specific binding constants for actinomycin D on DNA determined from footprinting studies. Biochemistry®, 31(4), 1046-1058.
Creative Commons License
This work is licensed under a Creative Commons Attribution 3.0 License.