Single-molecule assay reveals strand switching and enhanced processivity of UvrD

Abstract

DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3'-5' on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.

Fig. 1.

Experimental set-up. (A) Magnets (gray) exert an adjustable, constant force F on a bead tethered to the surface by a nicked DNA molecule. (B) Extension of a stretched dsDNA (blue), ssDNA (red), and a partially ds-ssDNA molecule (green) (normalized to the crystallographic length of the dsDNA). (C) Force–extension curves of ssDNA in the presence of increasing concentrations of UvrD ([ATP] = 500 μM). Within our working conditions ([UvrD] ≤ 1 nM), the change in extension of ssDNA is negligible.

Fig. 2.

Typical unwinding signals. At high forces, the extension of ssDNA is longer than dsDNA, and unwinding is observed as an increase in the molecule's extension (A and B): F = 35 pN; [UvrD] = 0.25 nM; [ATP] = 500 μM. (A) UH event (unwinding-rehybridization). (B) UZ event (unwinding-rezipping). (C) UvrD activity at F = 3 pN and [UvrD] = 50 nM. Unwinding is observed at low forces (F < 5 pN) as a decrease in the DNA's extension. Quick rehybridization (arrows) is still observed on dissociation of the enzyme. (D) Helicase activity observed at [UvrD] = 2 nM ([ATP] = 500 μM). At this UvrD concentration, there is enough binding of the enzyme to the ssDNA generated during an unwinding event to allow for successive rounds of unwinding, resulting in burst bunching. Notice also the simultaneous unwinding by two helicases. The arrow indicates when a second helicase has become active, doubling the unwinding rate. When one of the helicases dissociates, the rate of unwinding resumes its initial value.

Fig. 3.

Distributions of the unwinding and rezipping rates, lifetimes, and numbers of base pairs unwound at [ATP] = 500 μM. (A) The unwinding rate v_U (blue curve), rezipping rate v_Z (red curve), and the experimental noise v_N between bursts (green curve) are fitted to Gaussian distributions (dashed curves) of mean value 〈v_U〉 = 248 ± 3 bp/s, 〈v_Z〉 ≈ -298 ± 3 bp/s and 〈v_N〉 = -0.6 ± 0.7 bp/s and SD σ(v_U) = 74 bp/s, σ(v_Z) = 88 bp/s, and σ(v_N) = 32 bp/s. (B) Number of base pairs rezipped N_Z and rezipping lifetime τ_Z (Inset), fitted to Poisson distributions with mean value 〈N_Z〉 = 234 ± 13 bp and 〈τ_Z〉 = 0.73 ± 0.05 s. UH and UZ bursts give similar values of 〈v_U〉, 〈N_U〉, and 〈τ_U〉.(C) Number of base pairs unwound N_U and unwinding lifetime τ_U (Inset), fitted to Poisson distributions with mean value 〈N_U〉= 240 ± 14 bp and 〈τ_U〉= 0.98 ± 0.05 s. The error bars on the histograms represent the statistical error in the bins.

Fig. 4.

Rates, unwinding and rezipping extents and lifetimes as a function of ATP. (A) UvrD single-molecule unwinding rate 〈v_U〉 (blue), rezipping rate 〈v_Z〉 (red), and bulk ATP hydrolysis rate V_ATP (magenta) are fitted to the same MM kinetics. (B) The ratio 〈|v_Z|/v_U〉 is ATP-independent. The error on 〈v_z〉/v_u is only statistical. Because the value at 500 μM results from 288 points against typically 50 for the others, the associated error is much smaller and dominates the global mean value. (C) The mean unwinding extent N_U follows the same MM kinetics as the unwinding rate. (D) The unwinding lifetime 〈τ_U〉 is ATP-independent. Each point is the average over histograms obtained on typically five DNA molecules.

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.

Fig. 6.

The average power spectra of unwinding (blue, n = 438) and rezipping activity (red, n = 291) are fitted to δl² = δz〈v_U〉/2π²f² + b, leading to the same step size δz = 6 ± 1.5 bp. Experimental noise between activity bursts (green, n = 424) is shown for comparison.