Figure 2.
The frequency of LacI-binding events is reduced with the number of Cy3–TaMutS fluorophores on a mismatch DNA. a, an illustration of the mismatched DNA substrate. The 95-bp substrate contains a mismatch at 15T+ and a Cy5 fluorophore at the 24th nucleotide from the 5′-biotin bound to the smTIRF flow cell surface with the remaining end blocked by dig-antidig (supporting Table S1). Binding of Cy3–TaMutS to the mismatch results in a high FRET signal (E ∼ 0.8) that in the presence of ATP resolves into a sliding clamp with time-averaged FRET (E ∼ 0.3) as described previously (17). The binding of LacI and/or multiple sliding clamps alter the time-averaged FRET efficiency as shown below the middle and right molecules and as described in the text. b, representative trace showing the anti-correlation between Cy3–TaMutS (green) and Cy5–DNA (red) intensities caused by time-averaged FRET (blue) in the absence of a bound LacI (left) and in the presence of a bound LacI (right). Either natural or laser-intensity driven fluorophore photobleaching was recorded to determine the number of fluorophores bound to the mismatched DNA. Representative single step photobleaching of Cy3–TaMutS emission is shown. Binned histogram insets fit to normal distributions indicate mean FRET efficiency for one-fluorophore +LacI and −LacI with indicated number (N) of events. c, the frequency of LacI-bound DNA molecules. Total observations are shown above the data points from two separate experiments of a 40 μm × 80 μm field of view containing ∼60 well-resolved molecules. LacI-binding events were distinguished by their characteristic time-averaged FRET value and then binned with the number of photobleached fluorophores (see text).