Figure 1.

Observation of time-resolved Dpo4 binding to DNA. (A) A Cy3-labeled (blue) DNA primer-template surface immobilized onto a microscope slide is incubated with a solution containing Cy5-labeled (red) Dpo4. On binding, the polymerase acceptor fluoresces and the donor is quenched. (B) Shown at the top is the primer-template DNA sequence used for the single-molecule experiment shown below. The 8Cy3 notation indicates that there are eight nucleotides between the primer terminus and the T (blue) bearing the Cy3 fluorophore. Below, characteristic time trajectory of a single Dpo4-DNA complex. On binding, the donor fluorescence (blue) decreases with an anticorrelated increase in acceptor fluorescence (red). The apparent FRET (black line, bottom) for the same trajectory is shown. A single photobleaching event is observed at ∼62 s. An HMM fit of the FRET trace is shown in red. (C) On and off dwell time histograms (left and right, respectively) for 5 nM Dpo4 binding to 8Cy3. Both distributions were fit with mono-exponential decay functions. Off dwell times were measured as the time Dpo4 is bound to the DNA (t_on), whereas the on dwell times correspond to the time between binding events (t_off). (D) Transition density plot for Dpo4 binding to 8Cy3. The transition density plot displays all the FRET transitions calculated by the HMM fit. The initial FRET value is shown at the x-axis, and the final FRET value (after the transition) is shown at the y-axis.