Direct imaging of BRCA2 binding to RPA-coated ssDNA on single molecules of gapped λ DNA. (A) Schematic of experimental approach combining fluorescence microscopy, a microfluidic flow cell, and optical trapping, as well as the micromanipulation used to capture and image BRCA2 on individual DNA molecules. (B) Illustration of the gapped λ DNA generated through in vitro recombination of circular ssDNA with an engineered λ DNA. (C) Schematic of experimental protocol: Each molecule of gapped λ DNA was captured and micromanipulated between two beads held in separately controllable optical traps. The molecule was moved between solutions in a six-channel flow cell and successively incubated in a solution containing BRCA2. (D) Cartoon and microscopic image of a single molecule of gapped λ DNA (Left, stained with YOYO-1, cyan) that was destained and then successively incubated with BRCA2 (5 nM) plus α-BRCA2 and α-IgGAF546. Montage shows BRCA2 (magenta) binding to the gapped λ DNA at increasing time intervals. (E) Cartoon representation of the gapped λ DNA between two beads (Top) and histogram (Middle) of binding positions of BRCA2 (number of foci, N = 60). Each data point is also plotted as a single tick (Bottom) where the semi-transparent box represents the SE associated with assigning position owing to the optical resolution of the microscope. Gray bars represent the 10 to 90th percentile range of the 5′- and 3′-termined junctions (N = 98).