Figure 4.

Force-activation enables efficient determination of k_on for a helicase via a single-molecule assay. (A) An extension-versus-time trace prior to and immediately after force activation of the hairpin-unwinding substrate in the presence of RecQ^ΔH and saturating ATP. After a time t₁, unwinding of the newly created hairpin led to an increase in extension when measured under constant load (F = 8 pN). Note, the decrease in extension after activation matched the expected value (10 nm). Activation occurred in ∼0.5 s (orange bar). (B) Normalized histogram of t₁ at two different enzyme concentrations well below the K_D of RecQ^ΔH for ssDNA. An exponential fit to the histograms yielded the average time, denoted τ₁. All the data were acquired in a single day [N = 70 at 200 pM and N = 67 at 100 pM].