INTRODUCTIONThe 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).