Using the Chemistry of the Hydroxyl Radical to Determine Structural Details about DNA and Protein-DNA Complexes

The hydroxyl radical can be used as a highly sensitive chemical probe to study the structure of DNA (Tullius, 1989) and DNA complexed with proteins (Tullius & Dombroski, 1986; Tullius et al., 1987) or drug molecules (Churchill et al., 1990). The hydroxyl radical cleaves the DNA strand by abstracting a hydrogen atom from a deoxyribose along the DNA backbone. For structural studies, the major advantage of using the hydroxyl radical over large endonucleases, such as DNase I, stems from the radical being very small and highly reactive. In contrast to DNase I, the hydroxyl radical cleaves the DNA backbone non-specifically and at each position along the DNA molecule. The hydroxyl radical is generated by allowing the EDTA complex of iron (II) to react with hydrogen peroxide: [Fe (EDTA) ]2-+ H2O2 → [Fe (EDTA) ]1- + · OH + OH-. In this reaction an electron from iron (II) EDTA serves to reduce and break the 0-0 bond in hydrogen peroxide, giving as products iron (III) EDTA, the hydroxide ion, and the neutral hydroxyl radical. Sodium ascorbate is present to reduce the iron (III) product to iron (II) , thereby establishing a catalytic cycle and permitting low (micromolar) concentrations of iron (II) EDTA to be effective in cleaving DNA. A consequence of this scheme is that the concentrations of the three chemical species (iron (II) EDTA, hydrogen peroxide and sodium ascorbate) may be varied to optimise the generation of the hydroxyl radical under different solution conditions, e.g. to compensate for the presence of radical scavengers in the binding buffer of a protein-DNA complex. In this chapter, we will describe the basic protocol for hydroxyl radical cleavage of DNA, as well as protocols that allow one to measure the helical periodicity of DNA (Rhodes & Klug, 1980; Tullius & Dombroski, 1985; Hayes et al., 1990) to detect footprints (Tullius & Dombroski, 1986) , and to collect missing-contact information (Hayes & Tullius, 1989) for protein-DNA complexes.