Single-Stranded DNA Curtains for Single-Molecule Visualization of Rad51-ssDNA Filament Dynamics

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules

“…Due to space constraints, we were limited in the number of investigators we could highlight in this section and apologize to those who have contributed to the development and application of single-molecule assays but were not mentioned herein. Specific techniques that utilize TIRF microscopes to study protein-DNA complexes include DNA or protein tethering [39][40][41][42], DNA combing and curtains [43][44][45][46], DNA tightropes (via oblique angle fluorescence imaging) [47,48], as well as smFRET [40,[49][50][51][52][53] and TIRF/AFM systems [54][55][56]. While the examples mentioned below include data collected on both prism and objective-based TIRF instruments, in most cases, the prismTIRF microscope described above can be used for similarly designed experiments.…”

“…Homologous recombination (HR) is an important mechanism for the repair of doublestrand breaks (DSBs) and has been studied extensively via single-molecule techniques [43,[68][69][70][71][72][73][74][75][76]. This includes the ssDNA curtain assay developed by the Greene lab, which has provided new insight into HR regulation and the complex and heterogenous reaction intermediates that are involved [43,76]. One example is the multi-functional Mre11/Rad50/Nbs1 (MRN) complex, which initiates DSB repair by recognizing and resecting the free DNA ends at damaged sites.…”