Please cite as: CSH Protocols; 2007; doi:10.1101/pdb.prot4810

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Hydroxyl-Radical Footprinting

Michael Carey and Stephen T. Smale

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).


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