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footprinting

A group of techniques for identification of protein-binding sites on DNA. In dimethylsulphate (DMS) footprinting, for example, an end-labelled DNA fragment is partially methylated at guanidine residues with DMS, then exposed to the DNA-binding protein. The material is electrophoresed on a non-denaturing polyacrylamide gel to separate fragments that are methylated in the protein-binding sequence (and will no longer bind) from those that are methylated only elsewhere and will be less mobile due to the bound protein. The two gel bands are then eluted and cleaved with piperidine at the methylated sites. Comparison of the cleavage ladders of the two preparations will show unique bands due to methylation and cleavage at residues in the non-binding material that were essential for binding. In protection footprinting a DNA fragment with and without a binding protein is subjected to cleavage in non-binding areas, e.g. by partial methylation and cleavage by piperidine. The material is separated on a sequencing gel and the ladder of the protein-bound preparation will show a gap (the footprint) in its sequence where the protein has bound, compared with the ladder from 'naked' DNA. Protection footprinting may be applied to intact cells and compared with naked DNA in vitro as the control. In that case, ligand-mediated PCR is used to label and amplify desired areas of the DNA. Other methods of cleavage are by DNAse I and by hydroxyl radicals (OH•). (see also genetic footprinting)Lakin, N.D. (1993) in Transcription Factors: A Practical Approach (Latchman, D.S., ed.), pp. 27-47, Oxford University Press, Oxford

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