Mechanics and Single-Molecule Interrogation of DNA Recombination: Supplemental Video 7

A supplemental video from the 2016 review by Jason C. Bell and Stephen C. Kowalczykowski, "Mechanics and Single-Molecule Interrogation of DNA Recombination," from the Annual Review of Biochemistry.

Supplemental Video 7 Direct imaging of nucleation and growth of RecA on SSB-coated ssDNA using TIRF microscopy.

This video first shows a flow-extended single molecule of 3′-biotinylated λ ssDNA coated with a fluorescent protein, SSB^AF488. The molecule was tethered to a streptavidin-coated glass surface within a microfluidic flow cell and visualized using total internal reflection fluorescence microscopy. Solution flow is left to right. SSB^AF488 was exchanged with non-fluorescent SSB on the ssDNA in situ, and filament assembly was initiated by injecting fluorescein labeled RecA (350 nM) with nucleotide cofactor (ATPγS). Filament assembly proceeded primarily in the absence of flow or laser excitation, which were both used only during brief intermittent, time-lapsed imaging. Images were processed and rendered into a topographical intensity map for clarity. Published with permission from Reference 110.