Skip to main content
. 2002 May;46(5):1231–1239. doi: 10.1128/AAC.46.5.1231-1239.2002

FIG. 3.

FIG. 3.

Modulation of the helicase activity of WN virus NTPase/helicase by HMC-HO4. (A and B) Investigation of helicase activity as a function of increasing concentrations of HMC-HO4. The reaction took place in the absence (lanes 1 and 2) or in the presence (lanes 3 to 6) of the compound, which was added to the reaction mixture simultaneously with the enzyme. The concentration of HMC-HO4 was adjusted to 1 μM (lane 3), 10 μM (lane 4), 100 μM (lane 5), and 1 mM (lane 6). The substrate (lane 1) and the released strand were separated in a TBE-polyacrylamide gel and visualized by exposure of the dried gel to X-ray film for 20 h (A). Alternatively, the parts of the gels corresponding to the released strand were excised and the 32P radioactivity was quantified as described in Materials and Methods. The unwinding activity of the enzyme measured in the absence of HMC-HO4 was referred to as 100% (B). (C) Helicase activity of WN virus NTPase/helicase as a function of the duration of the interaction of HMC-HO4 with the DNA substrate. Aliquots of the DNA substrate were incubated in the presence of 20 μM HMC-HO4 or in the absence of the compound. At the indicated time points, the unwinding reaction was started by addition of the enzyme or the enzyme and HMC-HO4 as described above. The activity of the enzyme was determined as described above for panel B. The unwinding activity of the enzyme measured without the preincubated substrate was referred to as 100%. The inset demonstrates an expanded view of the region from 0 to 30 min of preincubation. (D) Effect of preincubation of the DNA substrate with HMC-HO4 on helicase activity as a function of increasing concentrations of the compound. Aliquots of the DNA substrate were incubated in the presence of increasing concentrations of HMC-HO4 or in the absence of the compound for 20 min (▾), 40 min (▪), and 90 min (⧫). Thereafter, the reaction was started by addition of the enzyme, and the unwinding activity was determined as described above for panel C. The results shown are representative of those for three independent experiments.