Mechanics and Single-Molecule Interrogation of DNA Recombination: Supplemental Video 8

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 8 Optical trapping and manipulation of single molecules of gapped λ DNA for direct imaging of RecA filament assembly.

This video first shows 1-μm streptavidin-coated polystyrene beads flowing through Channel 1 of a multichannel, microfluidic flow chamber and the subsequent isolation of two beads by a split-beam dual optical trap (Step 1). Solution flow is left to right. The beads are then transferred to Channel 2, which contains gapped λ DNA molecules comprising 8,155 nucleotides of SSB-coated ssDNA flanked by 21.08 and 24.59 kbp of YOYO-1 stained dsDNA; the gapped DNA is biotinylated at each of the molecule, and is captured in situ by binding to the streptavidin-coated beads (Step 2). The molecule is then transferred to a DNA-free Channel 3 where the distal end of the flow-extended molecule is captured by the other bead which is micromanipulated using a steerable mirror in line with one of the infrared laser beams (Step 3). The molecule is then rotated perpendicular to flow and imaged in buffer optimized for visualizing YOYO-1 stained dsDNA in Channel 4 (Step 4). The molecule is then transferred to Channel 5 containing Mg²⁺:ATPγS, which accelerates YOYO-1 dissociation (Step 6). The molecule was then successively incubated in reaction buffer containing fluorescein labeled RecA, Mg²⁺:ATPγS, and either RecOR or RecFOR and imaged in Channel 5 to measure the rates of nucleation and growth (Step 7). Published with permission from Reference 110.