DNase footprinting assay
DNase, or deoxyribonuclease, is a class of enzymes which digest DNA. The most common is the restriction enzyme DNase I, an endonuclease, which digests nucleic acids starting in the middle of the strand (as opposed to an exonuclease which must start at an end). A wide variety of DNase are known which differ in their substrate, specificities, chemical mechanisms and biological functions.
DNase Footprinting assay is a technique used in molecular biology by which it is possible to determine the location of a protein binding site on cellular DNA. It involves endonuclease treatment of an end labelled DNA fragment bound to a protein. Limited digestion produces fragments terminating everywhere except in the footprint region, which is protected from digestion.
The technique using restriction endonuclease (or restriction enzymes) uses an enzyme that cuts double-stranded DNA. The enzyme, without damaging the bases, makes two incisions – one through each of the phosphate backbones of the double helix. Provided the ends are complimentary, the chemical bonds that the enzymes sever can be reformed by other enzymes known as ligases, so that restriction fragments carved from different chromosomes or genes can be spliced together.
To investigate DNA-protein interactions, the cleavage pattern of isolated DNA is compared with the cleavage pattern of DNA in the presence of the commonly accepted binding protein. If the protein binds the DNA, the binding site is protected against restriction enzyme cleavage and fewer cleavage sites are found. From the position of the cleavage sites absent in the assay containing the binding protein, the position and extension of the binding site can be deduced.
Web Resources On DNase footprinting assay
Genome Research
DNA Protein Interactions
Book Resources On DNase footprinting assayMolecular Biology of the Cell: Problems Approach by John Wilson, Tim Hunt & Bruce Alberts
Methods in Enymology: Chromatin by Paul Wassarman and Alan Wolffe
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