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Quantitative DNase footprint titration: a tool for analyzing the energetics of protein–DNA interactions

Nature Protocols volume 3, pages 900–914 (2008)Cite this article

Abstract

A major goal in biomedical research is to determine the mechanisms responsible for gene regulation. However, the promoters and operators that control transcription are often complex in nature, containing multiple-binding sites with which DNA-binding proteins can interact cooperatively. Quantitative DNase footprint titration is one of the few techniques capable of resolving the microscopic binding affinities responsible for the macroscopic assembly process. Here, we present a step-by-step protocol for carrying out a footprint titration experiment. We then describe how to quantify the resultant images to generate individual-site binding curves. Finally, we derive basic equations for binding at each site and present an overview of the fitting process, applying it to the anticipated results. Users should anticipate that the footprinting experiment will take 3–5 d starting from DNA template isolation to image acquisition and quantitation.

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Figure 1: Schematic representation of the footprinting reaction.
Figure 2: Schematic layout of a DNA fragment used in DNase footprinting.
Figure 3: Quantitative footprint titration of a DNA promoter containing two identical, cooperatively interacting, binding sites (PRE2).
Figure 4: Transition curves generated from a footprint titration.
Figure 5: Schematic representation of a cooperative, two-site binding reaction.
Figure 6: Apparent fractional saturation of the reduced-valency template used in the global analysis of the PRE2 promoter.
Figure 7: Global analysis of the wild-type two site promoter and the reduced valency promoter.

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Acknowledgements

This work was supported by National Institutes of Health, USA grant R01-DK061933 to D.L.B.

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  1. Department of Pharmaceutical Sciences, University of Colorado Denver, Denver, 80262, Colorado, USA

    Keith D Connaghan-Jones, Amie D Moody & David L Bain

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Correspondence to David L Bain.

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Connaghan-Jones, K., Moody, A. & Bain, D. Quantitative DNase footprint titration: a tool for analyzing the energetics of protein–DNA interactions. Nat Protoc 3, 900–914 (2008). https://doi.org/10.1038/nprot.2008.53

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