Hydroxyl-Radical Footprinting
This protocol was adapted from “Theory, Characterization, and Modeling of DNA Binding by Regulatory Transcription Factors,” Chapter 13, in Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 1st edition, by Michael Carey and Stephen T. Smale. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.INTRODUCTION
The hydroxyl-radical footprinting methodology has wide applications to studying protein-DNA interactions, as well as structural perturbations (e.g., bending) that occur in DNA during protein binding. Hydroxyl radicals cleave DNA by abstracting a hydrogen atom from C4 of the sugar in the minor groove. Protein binding over the minor groove generally protects the sugar from cleavage. Because a hydroxyl radical molecule is small and thus is not subject to the same steric restrictions as other agents (such as DNase I), hydroxyl-radical footprinting can give detailed information on protein binding to the minor groove. In this protocol, the radical is generated by Fe(II) EDTA, which cleaves hydrogen peroxide into a hydroxyl radical and a hydroxide ion. The radical then cleaves the sugar in a diffusion limited reaction. Ascorbic acid is included to regenerate the active Fe(II).
