In terms of the mean dwell time and backstepping probability p −, the average unwinding velocity is given by v = d p + − p − τ, where d = 1 bp is the step size and p+ = 1 − p − is the forward stepping probability.
The backstepping probability p− depended inversely on p open.
Although 1-bp backsteps are more frequent as ATP is decreased, the backstepping probability remains non-zero at saturating ATP.
(In comparison, the 5-bp backstepping probability p−5 displayed hardly any dependence on sequence).
10.7554/eLife.00334.015 Figure 5. Sequence dependence of XPD processivity, backstepping probability, and dwell time.
At saturating ATP, kF = k+, the forward stepping rate, and k R = k − ∞, the backstepping rate, and the backstepping probability is given by the kinetic competition between the two.
(A ) Backstepping probability p −, (B ) Forward rate k F, and (C ) reverse rate k R vs p open at [ATP] = 500 μM.
For instance, in Figure 4E the backstepping probability was fit to 〈 p − 〉 = ∑ p − (p open ) ρ (p open ), where ρ(p open ) is the exact distribution of p open obtained in our measurements.
(For instance, the dwell time for 1-bp steps would be fit to a Michaelis-Menten equation, yielding two parameters, K M and k cat, etc) Similarly, the forward and backward rates (and the related backstepping probability, plotted in Figure 6A I) were measured as a function of sequence.
The reviewers also raised a specific point about the ATP dependence of the 5-bp backstep probability.
