1 - Quantitative DNase I Footprinting

Abstract

This chapter provides an overview on quantitative deoxyribonuclease I (DNase I) footprinting. DNase I footprinting was introduced by Schmitz and Galas to identify the DNA sequences that constitute binding sites for site-specific DNA-binding proteins. It has been used to detect changes in DNA structure and to measure the relative binding affinities of DNA-binding proteins. The unique value of footprinting as a quantitative titration technique for protein-DNA interactions is its ability to separately monitor the binding of protein to each specific site on the DNA. Further, the chapter focuses on the use of DNase I footprinting in vitro to yield data about specific binding sites and to obtain thermodynamic information that characterizes DNA-protein systems. One advantage of DNase I over chemical footprinting agents is that its enzymatic activity is specific to the DNA, so that degradation of the protein ligand is not a concern. A disadvantage is that its activity is somewhat sensitive to the DNA sequence, so that some regions are inefficiently cleaved even in the absence of bound protein ligand.