DNase I Footprinting

DNase I footprinting was developed by Galas and Schmitz in 1978 as a method to study the sequence-specific binding of proteins to DNA (). In the technique, a suitable uniquely end-labeled DNA fragment is allowed to interact with a given DNA-binding protein and then the complex 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 autoradiography 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. Because the DNase I molecule is relatively large as compared to other footprinting agents (see Chapters 5 and 6 on the use of hydroxy radicals and diethylpyrocarbonate), its attack on the DNA is relatively easily sterically hindered. 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. 1B). The xUBF interaction with the Xenopus ribosomal DNA enhancer can be easily detected by DNase I footprinting but has still not been detected by other footprinting techniques. Open image in new window Fig. 1. Examples of DNase I footprints. (A) Footprint (open box) of a chicken erythrocyte DNA binding factor on the promoter of the H5 gene (2) (figure kindly donated by A. Ruiz-Carrillo). (B) Interaction of the RNA polymerase I transcription xUBF with the tandemly repeated 60 and 81b.p. Xenopus ribosomal gene enhancers. Both (A) and (B) used 5′ end-labeled fragments. Minus and plus refer to naked and complexed DNA fragments, respectively, and G+A to the chemical sequence ladder.