Kinetics of DNA-protein association and dissociation by stopped-flow spectroscopy.

Abstract

The ability of proteins to recognize a specific nucleotide sequence or structural feature in the genome is central to DNA replication and repair as well as gene transcription. To understand how DNA-binding proteins accomplish this feat it is important to characterize how they efficiently recognize specific DNA sequences or various structural features in the milieu of a crowded nucleus containing 50mM basepairs of DNA. This minimally entails the determination of thermodynamic (equilibrium) parameters that characterize the relative affinities of DNA-binding proteins with their cognate and non-cognate DNA sequences of interest. Additionally, elucidating the kinetic parameters that govern DNA-protein association and dissociation can provide essential mechanistic insights into the underlying mechanisms, and can inform on how these processes can be controlled under varying cellular conditions. Unfortunately, the acquisition of kinetic data can appear intimidating and are usually not included in the characterization of most DNA binding proteins. Here we provide detailed theoretical considerations and practical, easy-to-follow protocols for using stopped-flow spectroscopy to study the kinetics of DNA-protein association and dissociation, using poly-(ADP ribose) polymerase 1 as a practical example.