Footprinting

Protein binding sites on DNA can be detected in vivo and in vitro by a technique termed “footprinting”. This depends on the principle that a bound protein molecule can protect DNA from cleavage or modification by a nuclease or a chemical reagent. The binding site is then defined by a region of reduced reactivity, which is determined by analyzing the products of the cleavage reaction on denaturing polyacrylamide gel. The footprint of the protein appears as a gap in the normal pattern of cleavage.

 In vitro DNase I is commonly used for enzymatic footprinting and dimethylsulfate and hydroxyl radicals are employed for chemical footprinting. DNase I targets the minor groove of DNA and is also sensitive to the local conformation of the bound DNA such that cleavage is enhanced where the minor groove is on the outside of DNA wrapped around the protein. Dimethylsulfate under footprinting conditions methylates the N7 position of guanine in the major groove and the N2 position of adenine in the minor groove. This methylation sensitizes the sugar-phosphate backbone to mild alkaline hydrolysis, enabling the sites of reaction to be determined. For hydroxyl radical footprinting the radicals are generated by the reaction of hydrogen peroxide with an Fe²⁺ –EDTA complex (the Fenton reaction) and they react with a backbone sugar resulting in the cleavage of the backbone. This reagent targets the minor groove and lacks sequence specificity, although it is sensitive to groove width, cleaving preferentially where the minor groove is widened. A similar type of reagent, Mn-porphyrin, has the opposite conformational selectivity, cleaving DNA where the minor groove is narrow.

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