(a–f) A single λ DNA molecule was tethered between two beads, then incubated in a SYCP3-containing or DNA-containing buffer while maintaining a transverse flow at 8 µm end-to-end separation. The flow drag prevents a random coil conformation of tethered DNA molecules and allows formation of large SYCP3-bridged regions between parallel flow-extended DNA molecules. Panels in the bottom row show possible interpretations for the arrangement of the DNA molecules bound by SYCP3. (a) After initial incubation, SYCP3 is observed to accumulate downstream along the DNA. No bridging is assumed to occur. (b) SYCP3 is subsequently concentrated in a bright spot, suggestive of local DNA-bridging. (c) After re-incubation in a DNA-containing buffer and subsequently in SYCP3-containing buffer, a bright spot (arrow), 8 µm downstream of the large SYCP3-DNA-cluster, indicates that SYCP3 is capable of bridging two or more separate DNA molecules. Additionally, the DNA to the left of the large SYCP3 cluster (box i) is uniformly coated with SYCP3, indicating a potentially extended bridged region between two DNA molecules. (d–f) Further SYCP3 incubation zips up the downstream tethered DNA over an increasingly long distance. (g) In a different experiment, multiple DNA molecules tethered and flow-stretched from a trapped bead appear uniformly bridged over their full parallel length. We estimated the number of SYCP3 molecules in boxes i-iv by calculating NSYCP3= (Ibox-Ibg)/(4Iref), where Ibox is the total photon count per box, Ibg is the total photon count in a box of the same size adjacent to the DNA-containing box, and Iref is the total photon count for a single fluorophore in a 2D image. Iref = A 2π (s/d)2 = 16.8, with A = 1.5 as the amplitude of a Gaussian fit for a single A555 dye (see Materials and methods), s = 100 nm the average standard deviation of the Gaussian fit and d = 75 nm the pixel size. The average SYCP3 footprint per box, calculated as the ratio of the length of the box over NSYCP3, was 4.1 nm, 3.6 nm, 3.4 nm, and 3.6 nm, for boxes i-iv respectively. 13 such analyses were performed, yielding an average footprint of 3.6 nm ± 0.3 nm (sd, N = 13). For comparison, in supposedly non-bridged conformations such as in panel a, we found an apparent average footprint of 9nm ±3 nm (sd, N = 7).