Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein–DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair.

Nucleotide excision repair (NER) is the primary mechanism for removal of ultraviolet light (UV)-induced DNA photoproducts and is mechanistically conserved across all kingdoms of life. Bacterial NER involves damage recognition by UvrA2 and UvrB, followed by UvrC-mediated incision either side of the lesion. Here, using a combination of in vitro and in vivo single-molecule studies we show that a UvrBC complex is capable of lesion identification in the absence of UvrA. Single-molecule analysis of eGFP-labelled UvrB and UvrC in living cells showed that UV damage caused these proteins to switch from cytoplasmic diffusion to stable complexes on DNA. Surprisingly, ectopic expression of UvrC in a uvrA deleted strain increased UV survival. These data provide evidence for a previously unrealized mechanism of survival that can occur through direct lesion recognition by a UvrBC complex.

Many protein interactions with DNA require specific sequences; however, how these sequences are located remains uncertain. DNA normally appears bundled in solution but, to study DNA-protein interactions, the DNA needs to be elongated. Using fluidics single DNA strands can be efficiently and rapidly elongated between beads immobilized on a microscope slide surface. Such "DNA tightropes" offer a valuable method to study protein search mechanisms. Real-time fluorescence imaging of these interactions provides quantitative descriptions of search mechanism at the single molecule level. In our lab, we use this method to study the complex process of nucleotide excision DNA repair to determine mechanisms of damage detection, lesion removal, and DNA excision.

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the viral transport from both the cargo and the ''gate'' (NPC) level in an unprecedented manner.