Kinetic simulation for pre-steady state DNA unwinding by NS3. A, kinetic scheme describing DNA unwinding by NS3. Two steps are needed to unwind a 30-bp DNA substrate. NS3 can dissociate from substrate at each step. A trapping strand is included in the simulation to prevent enzyme from rebinding to substrate. B, kinetic simulation for pre-steady state unwinding of 250 nm 15-nt/30-bp partial duplex DNA by 50 nm NS3 in the presence of a protein trap (75 μm nt polyuridine). Shown are results from kinetic simulations in which the unwinding rate constant, k_u, was held constant at 2.0 s^-1, whereas the dissociation rate constant, k_d, was set at 0 s^-1 (▪), 0.4 s^-1 (•), 1.0 s^-1 (○), 2.0 s^-1 (▴), 4.0 s^-1 (▵), and 8.0 s^-1 (□). C, single turnover conditions were used to measure DNA unwinding of a substrate containing 15 nt of ssDNA overhang and 30 bp (15 nt/30 bp). NS3 (500 nm) was incubated with the DNA substrate, and the unwinding reaction was initiated upon rapid mixing with ATP (5 mm) and MgCl₂ (10 mm). A protein trap (polyuridine, 75 μm nt) was introduced along with the ATP. Reactions were quenched with EDTA (200 mm), and products were examined by native gel electrophoresis. The data were fit to a two-step mechanism for unwinding using the program Scientist, resulting in an observed unwinding rate (k_u) of 1.03 ± 0.1 s^-1 and an amplitude of 0.8 ± 0.1 nm. D, DNA unwinding was conducted under the same conditions as in the kinetic simulation. NS3 (50 nm) was incubated with a 15-nt/30-bp DNA substrate (250 nm), followed by initiation of the unwinding reaction by the addition of ATP (5 mm) and MgCl₂ (10 mm). Product formation was measured in the presence (•) or absence (○) of a protein trap (75 μm nt polyuridine).

DNA unwinding under pre-steady state conditions reveals a low burst amplitude. A, the 15-nt/30-bp substrate (100 nm) was incubated with 50 nm (○), 100 nm (▴), 200 nm (▵), 300 nm (•), or 400 nm (□) NS3 prior to initiation of DNA unwinding upon mixing with ATP. The observed forward rate constants and amplitudes for product formation were determined by fitting the data to a two-step mechanism and are shown in Table 1. B, NS3 concentrations are plotted versus amplitudes from the data in A. The data were fit to a linear function resulting in a slope of 8.3.

Dynamic light scattering of NS3 supports an oligomeric structure. A, the size distribution histograms of NS3h as determined from dynamic light scattering. NS3h is shown to exist as a monomodal, monodisperse protein with a hydrodynamic radius of ∼3.9 nm. B, the size distribution histograms of NS3 as determined from dynamic light scattering. NS3 is shown to exist as a monomodal, polydisperse protein with a hydrodynamic radius of ∼29.4 nm.

Filtration through small pore filters removes the majority of NS3 but not NS3h. A, NS3 or NS3h was passed through a syringe tip filter of the indicated pore size, and the resulting filtrate was assayed for protein concentration by using a modified Bradford assay (A) and ATPase activity (B). NS3 concentration was 0.63 mg/ml prior to filtration. NS3 concentration was reduced to 0.06 and 0.03 mg/ml after passage through the 0.1- and 0.02-μm filters, respectively. NS3h concentration was 0.36 mg/ml prior to filtration. NS3h concentration was reduced to 0.35 and 0.32 mg/ml after passage through the 0.1- and 0.02-μm filters, respectively. B, polyuridine-stimulated ATPase activity was measured for NS3 and NS3h before and after filtration. NS3 (or NS3h) was incubated with polyuridine (100 μm), and measurements were made as described under “Experimental Procedures.” NS3-specific activity was 24 ± 1 s^-1 prior to filtration. NS3 activity was 1.9 ± 0.2 s^-1 and ∼0 after passage through the 0.1- and 0.02-μm filters, respectively. NS3h-specific activity was 49 ± 3 s^-1 prior to filtration. NS3h-specific activity was 34 ± 2 and 33 ± 2 s^-1 after passage through the 0.1- and 0.02-μm filters, respectively. C, NS3 was passed through a 0.02-μm filter, and the resulting solution was analyzed for DNA unwinding activity. No unwinding was observed (▾). An identical volume of unfiltered NS3 (250 nm) was incubated with the DNA substrate (15-nt/30-bp substrate, 2 nm), and the unwinding reaction was initiated upon rapid mixing with ATP (5 mm) and MgCl₂ (10 mm). A protein trap (polyuridine, 75 μm nt) was introduced along with the ATP. Reactions were quenched with EDTA (200 mm), and products were examined by native gel electrophoresis. Data were fit to a two-step mechanism, resulting in an observed unwinding rate of 1.4 ± 0.1 s^-1 and amplitude of 0.66 ± 0.05 nm. D, NS3h was passed through a 0.02-μm filter, and the resulting solution was analyzed for DNA unwinding activity (▴). An identical volume of unfiltered NS3h (1.0 μm) was analyzed for DNA unwinding activity (○). A double-push method was used for these experiments, because NS3h catalyzes spontaneous unwinding of the 15-nt/30-bp substrate in the absence of ATP at 37 °C. The first push mixed NS3h with the DNA, followed by a brief incubation period of 10 s. The second push rapidly mixed the enzyme with ATP (5 mm) and MgCl₂ (10 mm). A protein trap (polyuridine, 75 μm nt) was introduced along with the ATP. Reactions were quenched with EDTA placed in the receiving vial (200 mm), and products were examined by native gel electrophoresis. Data for NS3h were fit to a four-step mechanism, resulting in an observed unwinding rate constant of 2.6 ± 0.2 s^-1 and amplitude of 0.14 ± 0.01 nm for the unfiltered enzyme and observed rate constant of 2.3 ± 0.2 s^-1 and amplitude of 0.11 ± 0.01 nm for the filtered enzyme.

DNA unwinding under varying conditions with equimolar enzyme and substrate concentrations. NS3 (100 nm) was incubated with DNA (100 nm, 15-nt/30-bp substrate), followed by the addition of ATP and Mg⁺² to start the reaction. DNA unwinding was conducted under the following conditions. A, in the presence of 1 mm (•), 2.5 mm (▪), 5 mm (♦), and 7.5 mm (▴) ATP with 10 mm MgCl₂. B, in the presence of 1 mm (▴), 2.5 mm (♦), 5 mm (▪), and 10 mm (•) MgCl₂ with 5 mm ATP and 1 mm MgCl₂ with 1 mm ATP (▵). C, in the presence of 0% (•), 0.01% (▪), and 0.1% (▴) Tween 20 with 5 mm ATP and 10 mm MgCl₂. Data were fit to a linear function.

Time dependence of NS3 cross-linking using BS³ indicates oligomerization. A, silver stain of a SDS-PAGE (4–15% gradient) gel showing time dependence of cross-linking of NS3h. The experiment was carried out by adding 500 μm BS³ to a solution containing 2 μm NS3h in 50 mm MOPS-K⁺ (pH 7.0) and 10 mm NaCl, which had incubated at 37 °C for 30 min. Aliquots of the reaction mixture were removed at various times and quenched by adding 1 m glycine (pH 8.0). B, silver staining of a SDS-PAGE (4–15% gradient) gel showing time dependence of cross-linking of NS3 under the same condition as above for NS3h.

Size exclusion chromatography of NS3 supports an oligomeric structure. A, NS3 and NS3h (450 pmol) were injected onto a Bio-Sil SEC 250-5 column (Bio-Rad) in buffer containing 0.1 m potassium phosphate, pH 7.0, 10 mm NaCl. The standard curve (closed red diamonds) was prepared as described under “Experimental Procedures.” The red diamonds represent the elution times for standard proteins: thyroglobulin (670,000 Da), IgG (158,000 Da), ovalbumin (44,000 Da), and myoglobin (17,000 Da). NS3 (blue line) eluted from the column near the void volume, and NS3h (black line) eluted as a monomer of 50 kDa. B, NS3 and NS3h were analyzed as described in A except that the NaCl concentration was raised to 150 mm.