Fig. 5.

(A) Single helicase activity is observed only above 30 pN. ([UvrD] ≈ 0.25 nM, [ATP] = 500 μM, a low-pass filter at 1 Hz was applied to the time trace for clarity). (B) Above 30 pN (Right), the separated strands behind the helicase are slightly mismatched in the stressed strand as compared with the free one. This mismatch inhibits rehybridization as long as the enzyme binds to the fork and thus hinders nucleation. When UvrD dissociates, reannealing can proceed from the seed provided by the fork. Below 30 pN, the kinetic barrier preventing reannealing behind the helicase is lowered so the strands are able to rehybridize behind the helicase, preventing any observation of an unwinding signal. (C) Kinetic scheme of UvrD activity. E refers to free enzyme, D_n to free DNA with n·δz bp unwound. and denote the UvrD–DNA complexes in U and Z states respectively, with n·δz bp unwound. Dashed arrows indicate events that are due to resumption of the enzymatic activity by the same enzyme or by another one bound to the ssDNA fraction. Note that k_H corresponds to the rehybridization of 1 bp, in contrast with k_u and k_z, which correspond to steps of δz bp.