Fig. 1.

Single molecule TIRF time trajectories showing SSB pushing by Pif1. (A) 3′dT-Cy3-(dT)₁₄₀ DNA immobilized on the slide surface via a biotin-Neutravidin-biotin linkage displays only Cy3 fluorescence. (B) On addition of Cy5-SSB(A122C) (1 mM) and washing out free protein, anticorrelated Cy3 and Cy5 fluorescence fluctuations are observed indicating SSB diffusion along the ssDNA. (C) Addition of Pif1 (100 nM) to the 3′dT-Cy3-(dT)₁₄₀ DNA, followed by ATP (5 mM) and washing out free protein, results in repetitive Cy3 enhancement (PIFE) spikes. (D) Addition of Pif1 (100 nM) with ATP (5 mM) to the 3′dT-Cy3-(dT)₁₄₀ DNA prebound with Cy5-SSB results in a replacement of SSB diffusing FRET signals with Pif1 translocating PIFE signals and intermittent asymmetric FRET spikes reflecting Pif1 pushing of SSB in a 5′ to 3′ direction. 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, 100 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.