DNase I Footprinting
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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.
KeywordsGeiger Counter Nonspecific Competitor Pyridine Formate Standardize Vial Enrich Protein Fraction
- 5.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.Google Scholar
- 6.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.Google Scholar
- 8.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.Google Scholar