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Footprinting protein–DNA complexes using the hydroxyl radical

Nature Protocols volume 3, pages 1092–1100 (2008)Cite this article

Abstract

Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA–protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA–protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA–protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.

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Figure 1: Correspondence of the structure of a DNA–protein complex (right) and the hydroxyl radical footprint as imaged by an electrophoresis gel (left).
Figure 2: Hydroxyl radical footprint of the CUP2 protein bound to a 154-bp restriction fragment.
Figure 3: Details of the chemistry of the hydroxyl radical with the deoxyribose backbone of DNA.
Figure 4: Products of hydroxyl radical cleavage that result from the abstraction of a 4′-hydrogen atom from the deoxyribose sugar.

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Acknowledgements

We thank Dr. Wendy Dixon for producing the CUP2 footprint shown in Figure 2.

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  1. Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, 02215, Massachusetts, USA

    Swapan S Jain & Thomas D Tullius

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Correspondence to Thomas D Tullius.

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Jain, S., Tullius, T. Footprinting protein–DNA complexes using the hydroxyl radical. Nat Protoc 3, 1092–1100 (2008). https://doi.org/10.1038/nprot.2008.72

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