Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism

Abstract

Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork ( approximately 400 bp/s), its unwinding rate extrapolated to zero force is much slower ( approximately 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.

Fig. 1.

DNA unwinding by gp41. (A) Experimental setup. Two magnets exert a controlled force on a magnetic bead tethered to a single DNA hairpin. (B) gp41 activity on the 231-bp hairpin results in a series of well separated, transient DNA extension events. In the conditions used here ([ATP] = 5 mM; F = 10 pN), the vast majority of events result in the full unwinding of the duplex. (C and D) Two types of events are observed on the 231-bp substrate: the first type (C) displays a slowly rising edge caused by DNA unwinding and a fast falling edge corresponding to spontaneous DNA rehybridization upon gp41 dissociation; the second type (D) displays a slowly rising edge until the duplex is fully open followed by a slowly falling edge corresponding to rezipping or the fork closing in the protein's wake ([ATP] = 5 mM; F = 10 pN). Thin arrows indicate gp41 motion, and thick arrows indicate bead motion. (E and F) The two types of events also are observed at lower force ([ATP] = 5 mM; F = 6 pN). (E) Unwinding followed by rapid spontaneous reassociation of the two strands. (F) Unwinding followed by rezipping. The unwinding rate depends on the force (compare rising edge slopes of C and D versus E and F), whereas the rezipping rate is force-independent (compare the falling edge slope of F versus that of D).

Fig. 2.

Unwinding rate v_U and rezipping rate v_Z measured as a function of force at saturating ATP concentration. (A) The unwinding rate (black circles) displays a 10-fold increase as the force is increased from 3 to 11.5 pN. The rezipping rate (gray squares) does not display any significant variation over the force range explored ([ATP] = 5 mM, [gp41] = 100 nM monomer, 6.8-kbp substrate). (B) Events observed on the 231-bp substrate in the high force regime displaying four phases: (i) complete duplex unwinding by gp41; (ii) short extension plateau at the fully unfolded state; (iii) rapid, spontaneous rehybridization of the two DNA strands up to the helicase position; and (iv) gp41 rezipping with the fork closing in its wake. This type of event allows for the direct comparison of the rezipping rate (slope of phase iv) with the ssDNA translocation rate by gp41 on the stretched ssDNA (slope of the black dotted line; see text). F = 9 ± 1 pN, [ATP] = 5 mM, [gp41] = 100 nM monomer, 231-bp substrate. (C) Schematic representation of the event displayed in B. The ssDNA translocation rate is equal to δL/δt, or equivalent to the slope of the dashed line. (D) Rezipping rate distribution and ssDNA translocation rate distribution measured on events displayed in B. The histograms were fit to Gaussian distributions, yielding averages of 322 ± 17 bp/s (SD 92 bp/s) for the rezipping rate (gray) and 314 ± 15 bp/s (SD 83 bp/s) for the ssDNA translocation rate (black); F = 9 ± 1 pN, [ATP] = 5 mM, [gp41] = 100 nM monomer, 231-bp substrate; N = 43 events.

Fig. 3.

Force-averaged rezipping velocity. 〈∣v_Z∣〉 (ssDNA translocation velocity; see text) as a function of ATP concentration. The rezipping velocity obeys Michaelis–Menten kinetics with a maximum rate v_Z^max = 400 ± 10 bp/s and K_m = 1.1 ± 0.1 mM; data collected on the 6.8-kbp substrate (SI Text).

Fig. 4.

Proposed kinetic scheme. For clarity, the enzyme is drawn performing 1-bp steps; however, the step size is a free parameter of the model. (A) Model for ssDNA translocation includes reversible ATP binding followed by ATP hydrolysis and translocation by one step. The star denotes the ATP-bound enzyme. (B) Model for dsDNA unwinding. ATP binding and translocation steps have the same rate constants as in the ssDNA case (A). The enzyme can only perform the translocation step if the next n bp (the enzyme step size) is open. DNA opening and closing is modeled by the force-dependent rate constants α and β. If the enzyme is passive, these rates do not depend on the position of the enzyme relative to the fork. However, in the case of an active helicase, the presence of the enzyme at the fork destabilizes the junction; therefore, α and β depend both on the force exerted and on the relative distance of the helicase to the fork. Steps within the gray box (the force-dependent and translocation steps) can be combined in a unique step represented as k₂(F), where k₂ depends on the force, the step size of the helicase, and the type of unwinding mechanism (active versus passive).

Fig. 5.

Dependence of the unwinding rate v_U on the force F. (A) Unwinding rate versus force at various ATP concentrations. Circles, experimental data (N = 100 events typically, 6.8-kbp substrate; SI Text); solid lines, fit to the helicase kinetic model (see text). Global fitting of the data yields the number of base pairs opened in one enzymatic cycle as n = 1.4 ± 0.25 bp and the effective base-pairing energy at zero force as ΔG₀ − ΔG_Heli = 1.9 ± 0.25 k_BT. (B) Unwinding rate normalized to the ssDNA translocation rate versus force curves calculated for theoretical helicases varying in their level of double helix destabilization. Blue, 0% destabilization (a perfectly passive helicase); green, 50% destabilization; yellow, 90% destabilization; red, 99% destabilization. Extent of destabilization is given as the ratio of the energy input by the helicase divided by the average base-pairing energy in the absence of force: ΔG_Heli/ΔG₀, (where ΔG₀ = 1.95 k_BT). Except for ΔG_Heli, the rates and step size are equal to those found for gp41 in our experiments; [ATP] = 5 mM. As the energy input increases, the unwinding rate reaches the ssDNA translocation rate (high force plateau observed for all curves) more rapidly. At F = 0, the unwinding rate of an optimally active helicase would be very close to the ssDNA translocation rate.