Functional Studies of DNA-Protein Interactions Using FRET Techniques

Abstract

Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of protein-DNA interactions.

Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy

“…Analyses of protein–DNA interactions on the basis of the FRET phenomenon are widely used in biochemistry [ 35 ]. Interactions between the enzyme and FRET-labeled damaged DNA cause a bending of the substrate duplex, resulting in fluctuation of the distance between the donor and acceptor and changes in FRET efficiency.…”

Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain

“…APE1 regulatory mechanisms probably imply dynamic variations in the relative positions of the N-terminal tail with respect to the globular domain and of both domains with respect to the DNA. Another methodological approach to evaluate intra-and intermolecular conformational rearrangements that may underpin APE1 function and regulation is Förster resonance energy transfer (FRET), a fluorescence spectroscopy technique sensitive to the distances between two fluorophores (donor and acceptor of the transference) [106]. In particular, novel single-molecule methodologies provide us with the spatial and temporal resolution to follow molecular movements that could be hidden in the average of sample populations [107] and can be applied to proteins containing intrinsically disordered regions [108], such as the N-terminal tail of APE1.…”

Nanoscale surface structures of DNA bound to Deinococcus radiodurans HU unveiled by atomic force microscopy

“…A wide range of biophysical approaches can be used to probe the conformation of isolated DNA molecules and DNA-protein assemblies, including single-molecule techniques such as molecular tweezers, 1 electron microscopy (EM), 2 atomic force microscopy (AFM), 3 and ensemble measurements such as electrophoresis 4 or Förster resonance energy transfer (FRET) for example. 5 AFM imaging has been widely applied to many DNA-based systems. It has successfully revealed configurations of free DNAs, 6,7 supercoiled plasmids, 8,9 and that of DNA-protein and DNA-polymer assemblies.…”