Figure 1.

Unwinding of 37:22-mer partial duplex by NS3 and NS3h in the presence of heparin (used as a protein trap) under conditions of enzyme concentration in excess of DNA substrate concentration. A “double-mixing” experimental protocol was performed as described in the text. Unwinding products were resolved on 20% polyacrylamide gel and visualized using a PhosphorImager and quantitated by using ImageQuant software. (A) Representative gel image showing unwinding of 2 nM partial duplex by 500 nM NS3. The blank sample (B) shows the partial duplex DNA substrate prior to unwinding, and sample (H) shows the DNA after heating to 95 °C for ten min. (B) Representative gel image showing unwinding of 2 nM partial duplex by 500 nM NS3h. (C) To determine the efficiency of heparin as a protein trap, the partial duplex substrate (2 nM) was mixed with heparin (4 mg/ml) prior to initiating the reaction upon mixing with NS3h (500 nM). Little or no unwinding was observed. (D) Fraction of product formation for unwinding of 2 nM partial duplex by 500 nM NS3 (), 500 nM NS3h (), and 500 nM NS3h under conditions where heparin and partial duplex DNA were mixed together prior to initiation of the reaction by addition of enzyme. Enzyme-catalyzed unwinding was fit to a single exponential followed by a steady state rate resulting in amplitudes of 0.19 ± .03 and 0.17 ± .01 nM, rate constants of 0.62 ± 0.3 and 0.61 ± 0.1 s⁻¹, and steady state rates of 0.005 ± .001 and 0.004 ± 0.001 nM/s for NS3 and NS3h, respectively. The control experiment in which heparin was added to the DNA substrate prior to addition of NS3h was fit to a linear equation resulting in a rate of 0.004 ± 0.001 nM/s.().