Protein–DNA Interactions: Techniques Used

Mark A Strauch, University of Maryland, Baltimore, Maryland, USA

Published online: April 2001

DOI: 10.1038/npg.els.0003128

Abstract

A plethora of biochemical and biophysical techniques are used to investigate various aspects of protein–DNA interactions. Some of the more common and useful of these are filter binding, the gel mobility shift assay, a large group of related techniques broadly categorized as footprinting, high‐resolution microscopy and spectroscopy.

Keywords: protein–DNA complexes; techniques; DNA‐binding proteins

Figure 1.

Filter binding and gel mobility shift assays. For illustrative purposes, a hypothetical set of interactions between a binding protein (P) and a DNA fragment containing two separate and independent binding sites (x and y) is shown. Separation of a mixture of unbound (UNB) DNA and each of the three possible bound forms, by (a) filter binding and (b) gel mobility shift, is depicted. Note that filter binding does not discriminate between the different bound forms, but the gel mobility shift is able to fractionate the doubly‐bound complexes away from the singly‐bound species (in this case it is assumed that the two distinct singly‐bound species cannot be distinguished solely on the basis of gel mobility).
Figure 2.

DNAase I footprinting: an example of the protection class of footprinting assays. Unbound and protein‐bound DNA fragments (labelled (asterisk) at one end on one strand) are treated with limiting amounts of DNAase I (no more than one cleavage per molecule). The digestion reactions are fractionated on a denaturing polyacrylamide gel. Protein bound to DNA protects areas from DNAase I attack. The absence of bands corresponding to digestion products generated by cleavage in these areas defines the protein‐binding site (the ‘footprint’).
Figure 3.

Footprinting interference assay. End‐labelled DNA (asterisk) is subjected to limited chemical modification before exposure to the binding protein. The binding reaction is fractionated via a gel mobility shift assay to separate bound (B) from unbound (UNB) species. The DNA in the bound (retarded) band will either be unmodified or contain modifications at positions not affecting binding. The DNA in the unbound band will be comprised of molecules in which modification has occurred at positions affecting interaction with the protein. The DNA from both bands is isolated, subjected to strand cleavage at positions adjacent to the site of modification, and fractionated on a denaturing polyacrylamide gel. Positions which, when modified, affect protein binding will produce corresponding cleavage products only in the sample from the unbound species.
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References

Bogenhagen DF (1993) Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA. Molecular and Cellular Biology 13: 5149–5158.

Bustamante C and Rivetti C (1996) Visualizing protein–nucleic acid interactions on a large scale with the scanning force microscope. Annual Review of Biophysics and Biomolecular Structure 25: 395–429.

Darst SA, Polyakov A, Richter C and Zhang G (1998) Structural studies of Escherichia coli RNA polymerase. Cold Spring Harbor Symposia on Quantitative Biology 63: 269–276

Heyduk E and Heyduk T (1994) Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach. Biochemistry 33: 9643–9650.

Lane D, Prentki P and Chandler M (1992) Use of gel retardation to analyze protein–nucleic acid interactions. Microbiological Reviews 56: 509–528.

Miller DP (1996) Fluorescence studies of DNA and RNA structure and dynamics. Current Opinion in Structural Biology 6: 322–326.

Schuck P (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of the interactions between biological macromolecules. Annual Review of Biophysics and Biomolecular Structure 26: 541–566.

Taylor I and Kneale GG (1994) A competition assay for DNA binding using the fluorescent probe ANS. In: Kneale GG (ed.) DNA–Protein Interactions: Principles and Protocols, pp. 327–337. Totowa, NJ: Humana Press.

Thomas GJ Jr (1999) Raman spectroscopy of protein and nucleic acid assemblies. Annual Review of Biophysics and Biomolecular Structure 28: 1–27.

Thresher R and Griffith J (1992) Electron microscopic visualization of DNA and DNA–protein complexes as adjunct to biochemical studies. Methods in Enzymology 211: 481–490.

Wong I and Lohman TM (1993) A double filter method for nitrocellulose‐filter binding: application to protein–nucleic acid interactions. Proceedings of the National Academy of Sciences of the USA 90: 5428–5432.

Further Reading

Jost JP and Saluz HP (eds) (1991) A Laboratory Guide to In Vitro Studies of Protein–DNA Interactions. Basel: Birkhäuser.

Kneale GG (ed.) (1994) DNA–Protein Interactions: Principles and Protocols. Totowa, NJ: Humana Press.

Revzin A (ed.) (1993) Footprinting of Nucleic Acid–Protein Complexes. San Diego: Academic Press.

Sauer RT (ed.) (1991) Protein–DNA interactions. Methods in Enzymology 208.

Related Sub-Topics:

Biomolecular Interactions | Nuclear Magnetic Resonance | Nucleic Acids: Structure, Function, Metabolism | Protein Structure | Techniques and Tools in Molecular Biology | Biochemical and Biophysical Techniques | Proteins: Structure, Function, Metabolism | Electron Microscopy
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Article Title: Protein–DNA Interactions: Techniques Used
 
How to Cite close
Strauch, Mark A(Apr 2001) Protein–DNA Interactions: Techniques Used. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003128]