Characterization of protein-DNA complexes by affinity cleaving

Abstract

High-resolution crystallographic views of protein-DNA complexes reveal the structural complexity of protein-DNA interactions. The combination of direct protein-DNA contacts mediated by multiple hydrogen bonds and sequence-dependent DNA conformational effects limits our ability to make detailed structural predictions, even if a new DNA-binding protein can be assigned to a structural class such as helix-turn-helix, double-barreled helix, zinc-binding finger, or scissor grip-leucine zipper. In the absence of high-resolution crystallographic and nuclear magnetic resonance (NMR) data, solution methods such as affinity cleaving can be used to characterize the topology of protein-DNA complexes and correlate sequence similarities with known structural classes. The conversion of a sequence-specific DNA-binding protein into a sequence-specific DNA-cleaving protein by covalent attachment of the iron chelator, ethylenediaminetetraacetic acid (EDTA), to a specific amino acid residue creates a class of hybrid affinity-cleaving proteins that are available through chemical synthesis. Moreover, a structural domain consisting of naturally occurring amino acids that binds transition metals and oxidatively cleaves DNA extends this method to recombinant methods for protein synthesis.