Figure 4.

Dwell Time of Pauses with Lower dTTP Concentration and Backward Movements

(A) Percentage of unwound molecules as a function of [dTTP].

(B) Unwinding time from high FRET to low FRET as a function of [dTTP].

(C) A step-finding algorithm was used to measure FRET values and dwell times of each pause for a single molecule. Experiments were performed using 100 μM dTTP on an 80% GC substrate.

(D) Gamma distribution fitting of the collected dwell times at each pause for >50 molecules.

(E) Cy3 and Cy5 intensity trace during unwinding on an 80% GC sequence, depicting backward movements by the T7 helicase during unwinding at 100 μM [dTTP] (top left panel), and Cy3 and Cy5 intensity trace displaying unwinding without backward movements at 1 mM [dTTP] (bottom left panel). The lines indicate the areas selected to calculate cross-correlation. Top and bottom right panels: calculated FRET efficiency versus time for the fluorescence intensity trace. Smoothed curves that were used for cross-correlation analysis are depicted in blue (see Supplemental Experimental Procedures).

(F) Percentage of backward-movement traces as a function of [dTTP].

(G) Average cross-correlation curve from 50 molecules for 100 μM (left panel) and 1 mM dTTP (right panel).

See also Figure S2.