Probing DNA-Protein Interactions with Atomic Force Microscopy
Large, multi-component protein assemblies are involved in many DNA transactions such as recombination, replication, transcription, and repair. In order to progress in the understanding of different key steps of these mechanisms, it is imperative to analyze the structure of the DNA-protein complexes involved and the dynamic interactions that govern their assembly, function and disassembly. These complexes are usually studied in vitro using a combination of biochemical and biophysical methods. A direct visualization technique such as AFM imaging is unique in that it allows one to characterize mechanisms involved in DNA-protein recognition and DNA-protein complex formation in different conditions (air and liquids of varying composition) with nanometer resolution. By comparison, other single molecule techniques such as optical or magnetic tweezers give indirect measurements based on the mechanical properties of single DNA molecules, and place severe restrictions on DNA size. Fluorescence microscopy can be used in conjunction with such approaches, but requires staining of either the DNA or the proteins and has relatively low resolution (about 200 nanometers). Owing to successive improvements to sample preparation and experimental methodology –along with instrumentation progress, mostnotably the invention of TappingModeand the ability to operate in fluid– AFM has indeed become a powerfuland complementary tool to probeDNA-protein interactions at thesingle molecule level. The purpose of this application note is mainlyto illustrate the potentialities of AFM imaging in the analysis of the architecture and dynamics of DNA-protein complexes, while introducing readers to the issue of DNA-protein complex adsorption, key for rationalizing sample preparation.
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