Abstract
DNase I footprinting was developed by Galas and Schmitz in 1978 as a method to study the sequence-specific binding of proteins to DNA (1). In this technique a suitable uniquely end-labeled DNA fragment is allowed to interact with a given DNA-binding protein and then the complex is partially digested with DNase I. The bound protein protects the region of the DNA with which it interacts from attack by the DNase. Subsequent molecular weight analysis of the degraded DNA by electrophoresis and auto-radiography identifies the region of protection as a gap in the otherwise continuous background of digestion products (for examples, see Fig. 1). The technique can be used to determine the site of interaction of most sequence-specific DNA-binding proteins but has been most extensively applied to the study of transcription factors. Since the DNase I molecule is relatively large compared to other footprinting agents, its attack on the DNA is more readily prevented by steric hindrance. Thus DNase I footprinting is the most likely of all the footprinting techniques to detect a specific DNA-protein interaction. This is clearly demonstrated by our studies on the transcription factor xUBF (see Fig. 1 B). The xUBF interaction with the Xenopus ribosomal DNA enhancer can be easily detected by DNase I footprinting but has not yet been detected by other footprinting techniques.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schmitz, A. and Galas, D. J. (1978) DNase I footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5, 3157–3170.
Rousseau, S., Renaud, J., and Ruiz-Carrillo, A. (1989) Basal expression of the histone H5 gene is controlled by positive and negative cis-acting sequences. Nucleic Acids Res. 17, 7495–7511.
Suck, D., Lahm, A., and Oefner, C. (1988) Structure refined to 2 Å of a nicked DNA octanucleotide complex with DNase I. Nature 332, 464–468.
Drew, H. R. (1984) Structural specificities of five commonly used DNA nucleases. J. Mol. Biol. 176, 535–557.
Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages, in Methods in Enzymology, vol. 65 (Grossman, L. and Moldave, K., eds.), Academic, New York, pp. 499–560.
Current Protocols in Molecular Biology, Chapter 3 (1991) (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Smith, S. A., and Struhl, K., eds.), Greene and Wiley-Interscience, New York.
Walker, P. and Reeder, R. H. (1988) The Xenopus laevis ribosomal gene promoter contains a binding site for nuclear factor-1. Nucleic Acids Res. 16, 10,657–10,668.
Brenowitz, M., Senear, D. F., and Kingston, R. E. (1991) DNase footprint analysis of protein-DNA binding, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. E., Smith, S. A., and Struhl, K., eds.), Greene and Wiley-Interscience, New York, pp. 12.4.1–12.4.11.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Leblanc, B., Moss, T. (2000). DNase I Footprinting. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:729
Download citation
DOI: https://doi.org/10.1385/1-59259-038-1:729
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-459-4
Online ISBN: 978-1-59259-038-4
eBook Packages: Springer Book Archive