Chemical Protection and Premodification-Binding Interference for the Identification of Phosphate and Base-Specific Contacts in Protein-DNA Complexes

Abstract

The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and base-specific groups. Various (in-gel) footprinting methods (for more information, see Chapter 11 ) allow the identification of the global-binding region but do not provide details on the contribution to complex formation of individual sequence-specific constituents of the DNA-binding site. Here, we describe how various chemicals can be used to randomly and sparingly modify specific bases or phosphates and allow the identification of those residues that are specifically protected against modification upon protein binding (protection studies) or interfere with complex formation when modified or removed prior to protein binding (premodification-binding interference). Each one of these complementary approaches has its advantages and shortcomings and results have to be interpreted with caution, having in mind the precise chemistry of the modification. However, used in combination, these methods provide an accurate and high-resolution image of the protein-DNA contacts.

Keywords: Denaturing gel electrophoresis; Depurination; Depyrimidation; Gel retardation; Missing contact probing; Phosphate ethylation; Protein–DNA interactions; Purine methylation; Thymine oxidation; Uracil substitution.