Structure-based mutagenesis study of hepatitis C virus NS3 helicase - PubMed
Characterization of duplex nucleic acid unwinding and ssRNA binding activities of the wt HCV NS3 helicase. Standard helicase unwinding reactions (reaction volume, 20 μl) with the 3′-tailed double-stranded RNA-DNA hybrid substrate were carried out as described in the Materials and Methods section unless noted otherwise below. Effects of various parameters were examined as follows. (A) For characterization of the effect of enzyme concentration, 0.1, 0.3, 1, or 3 nM enzyme was incubated with 5 nM duplex substrate at 37°C for 0, 2.5, 5, 10, 20, 30, or 60 min. (B) For ATP 0.3 nM enzyme was incubated with 5 nM duplex substrate for 20 min at 37°C in the presence of 0.04, 0.06, 0.08, 0.1, 0.14, 0.2, 0.5, 1, or 5 mM ATP (the sodium salt form). Standard ssRNA binding reactions (reaction volume, 40 μl) with the 32P-labeled 34-nucleotide ssRNA were carried out as described in the Materials and Methods unless noted otherwise below. Effects of various parameters were examined as follows. (C) For examination of the effect of enzyme concentration, 0.3125, 0.625, 1.25, 1.875, 2.5, 3.75, 5, 6.25, 7.5, 8.75, 10, 12.5, 18.75, 25, 37.5, or 50 nM enzyme was incubated with 5 nM 32P-labeled ssRNA substrate at 30°C for 15 min. (D) For examination of the effect of the dissociation of ssRNA from the HCV helicase, 6.25 nM enzyme was incubated with 5 nM 32P-labeled ssRNA for 15 min at 30°C, and then 250 nM 3H-labeled ssRNA was added to the reaction mixture and incubated at 30°C for 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, or 60 additional min. The amount of 32P-labeled ssRNA which was bound to HCV NS3 helicase was shown.