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

Homologous recombination (HR) is a highly conserved DNA repair pathway required for the accurate repair of DNA double-stranded breaks. DNA recombination is catalyzed by the RecA/Rad51 family of proteins, which are conserved from bacteria to humans. The key intermediate catalyzing DNA recombination is the presynaptic complex (PSC), which is a helical filament comprised of Rad51-bound single-stranded DNA (ssDNA). Multiple cellular factors either promote or downregulate PSC activity, and a fine balance between such regulators is required for the proper regulation of HR and maintenance of genomic integrity. However, dissecting the complex mechanisms regulating PSC activity has been a challenge using traditional ensemble methods due to the transient and dynamic nature of recombination intermediates. We have developed a single-molecule assay called ssDNA curtains that allows us to visualize individual DNA intermediates in real-time, using total internal reflection microscopy (TIRFM). This assay has allowed us to study many aspects of HR regulation that involve complex and heterogenous reaction intermediates. Here we describe the procedure for a basic ssDNA curtain assay to study PSC filament dynamics, and explain how to process and analyze the resulting data.