FIG. 2.

Determination of NS1 helicase directionality. The preparation and 3′-end labeling of helicase substrates were done as described in Materials and Methods. Lanes I represent the circular helicase substrate before ClaI digestion, diagrammed on the upper right, which serve as a positive control. Lanes II contained the ClaI-digested helicase substrate, diagrammed on the lower right, used to determine directionality. As indicated at the top of the gel, the different helicase substrates were incubated with increasing amounts of GST-NS1 (10, 30, or 90 ng) or purified SV40 large TAg (25 and 75 ng). Helicase assays were carried out as described in Materials and Methods. A, 341-base HaeIII fragment derived from double-stranded M13 DNA; B and C, linear substrate with 143- (C) and 202-bp (B) duplexes connected by a stretch of single-stranded M13 DNA in which each of the 3′ ends is labeled (directional substrate).