Fig. S7.

Representative smTIRF time trajectories for SSB pushing by Rep. (A) 5′-Cy3-dT₁₄₀ DNA immobilized on the slide surface via a biotin-Neutravidin-biotin linkage displays only Cy3 fluorescence similar to the 3′-Cy3 DNA substrate. (B) Introduction of 100 nM Rep and 5 mM ATP results in repetitive Cy3 PIFE fluctuations. (C) Introduction of 100 nM Rep with 5 mM ATP to a surface with Cy5-SSB bound to 5′-Cy3-dT₁₄₀ DNA results in the loss of fluctuating FRET signals reflecting random SSB diffusion with only fluctuating Cy3 PIFE signals reflecting Rep translocation. Asymmetric saw-toothed anticorrelated FRET spikes are observed in 22% of molecules still displaying Cy5 emission. Green, Cy3 fluorescence; red, Cy5 fluorescence; blue, FRET efficiency calculated from the Cy3 and Cy5 signals. Solution conditions: 30 mM Tris⋅HCl, pH 8.1, 20 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA, 0.5% (wt/vol) dextrose, 3 mM Trolox, 1 mg/mL glucose oxidase, and 0.4 mg/mL catalase, 25 °C.