title: DNase I footprinting.
abstract: DNase I footprinting has found a wide following for both identifying and characterizing DNA-protein interactions, particularly because of its simplicity. The concept is that a partial digestion by DNase I of a uniquely (32)P-end-labeled fragment will generate a ladder of fragments, whose mobilities on a denaturing acrylamide gel and whose positions in a subsequent autoradiograph will represent the distance from the end label to the points of cleavage. Bound protein prevents binding of DNase I in and around its binding site and thus generates a "footprint" in the cleavage ladder. The distance from the end label to the edges of the footprint represents the position of the protein-binding site on the DNA fragment. The position of the binding site can be determined by electrophoresing a DNA sequencing ladder alongside the footprint. DNase I cannot bind directly adjacent to a DNA-bound protein because of steric hindrance. Hence, the footprint gives a broad indication of the binding site, generally 8-10 base pairs (bp) larger than the site itself.
authors: Michael F
authors: MF
authors: Carey
authors: Craig L
authors: CL
authors: Peterson
authors: Stephen T
authors: ST
authors: Smale
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D010761:Phosphorus Radioisotopes / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D010761:Phosphorus Radioisotopes; D004247:DNA; D003850:Deoxyribonuclease I