Fig. 4: Catenation of circular dsDNA around tethered ssDNA by TRR.

a Representative FD-curves (from N ≥ 20) of TRR-ssDNA before (green) and after (blue) incubation in high-salt buffer. For reference, the FD-curve of bare ssDNA is shown in orange. Arrows depict the changes in end-to-end distance: TRR binding in standard buffer results in ~49% lengthening of the ssDNA, which was reversed by ~−38% after moving the substrate into high-salt buffer due to TRR unbinding. b Representative mCherry fluorescence images (from N ≥ 20) of TRR-ssDNA before (top) and after (bottom) incubation in high-salt buffer. The scale bar represents 5 µm, and applies to all snapshots. c Representative intercalator fluorescence images (left; from N ≥ 10) and schematic representations (right) of circular dsT-DNA catenated around tethered TRR-ssDNA after incubation in high-salt buffer under different buffer flow conditions. The applied flow helps visualize the high mobility of the dsT-DNA. d Representative fluorescence images (from N = 3) of TRR-ssDNA (mCherry fluorescence, top) bound by linear dsT-DNA (intercalator staining, centre), recorded in standard buffer. A schematic representation of the interaction is also shown (bottom). e Representative fluorescence images (from N = 3) of TRR-ssDNA (mCherry fluorescence, top) bound by residual linear dsT-DNA (intercalator staining, bottom two images), recorded in high-salt buffer (same molecule as in panel (d)). The two bottom images were obtained several seconds apart and show a similar (albeit weak) fluorescence intensity pattern, suggesting that the dsT-DNA was bound to residual TRR that remained on the ssDNA. Note that when incubating in high-salt buffer, the tension was reduced to <1 pN in order promote protein unbinding. Source data are provided as a Source Data file.