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. 2016 May 16;113(22):6194–6199. doi: 10.1073/pnas.1602878113

Fig. S4.

Fig. S4.

Pif1-induced displacement of Cy5-SSB from 3′-dT-Cy3-(dT)70 DNA is ATP dependent. (A) FRET histograms for 3′-dT-Cy3-(dT)70 DNA only (green line), after incubation with 1 nM Cy5-SSB and subsequent washing away of unbound protein (red line), addition of 100 nM ScPif1 to Cy5-SSB/3′-dT-Cy3-(dT)70 DNA (black line), and addition of 100 nM ScPif1 with 50 μM ATP to Cy5-SSB/3′-dT-Cy3-(dT)70 DNA (blue line). n = 200 molecules for each condition. (B) Percentage of each type of single molecule behavior observed for each condition described in A. Cy3 DNA emission only (black columns) had only stable Cy3 emission with no enhancement spikes or Cy5 emission observed. Cy5-SSB/Cy3-DNA FRET (red columns) displayed anticorrelated Cy3 and Cy5 emission. The repetitive Cy3 enhancement (green columns) exhibited regularly spaced saw-tooth spikes of enhanced Cy3 emission (PIFE). Push-off event (blue column) indicates molecules that show a high FRET signal that disappears in a near step function and is replaced by repetitive Cy3 enhancement (PIFE).