chapter  32
2 Pages

DNase I Footprinting

The fragment containing the promoter sequence-and labelled radioactively at one of its ends-is hybridized with nuclear proteic extracts as before (see Profile 31). Then the combination is subjected to a moderated treatment with DNase I, which is an endonuclease that can cleave phosphodiester bonds within DNA chains of only one of the two strands. The result is a collection of fragments of different sizes, some of which are labelled at one end. The presence of a proteic complex conceals a possible site of restriction by DNase I and prevents the action of this enzyme. The products of digestion are then separated by electrophoresis on acrylamide gel with a high separating power (of the same type as sequence gels), then detected by autoradiography. When the bands obtained for a fragment of cleaved DNA are compared in the presence or absence of proteic factors, a region is revealed without a corresponding band at the site of fixation of the proteic factor, which by its linkage with DNA has protected the site of restriction with DNase I. In realizing a sequencing of the fragment studied and causing the migration of products of reaction in parallel with those obtained during an experiment in footprinting with DNase I, it will be possible to determine the sequence of the fixation site of the proteic factor.