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

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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:

Biochemical and Biophysical Techniques | Nucleic Acids: Structure, Function, Metabolism | Protein Structure | Biomolecular Interactions | Nuclear Magnetic Resonance | Techniques and Tools in Molecular Biology | 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]