Fig 3.

Single nucleotide incorporation. (a) Single nucleotide incorporation by wild-type BVDV NS5B and its mutant derivatives, using duplex A (Table 1) as the template primer pair. NS5B (2.5 μM) and the RNA duplex (0.1 μM) were mixed under standard reaction conditions with 500 μM ATP in the presence of 5 mM MgCl₂ and incubated at 37°C for 30 min. Samples (1.5 μl) were withdrawn at various time points and quenched by adding denaturing buffer prior to PAGE analysis. (b) Single nucleotide incorporation by wild-type BVDV NS5B under the same reaction conditions as those described for panel A, but in the presence of MnCl₂. The results shown are representative; each assay was reproduced a minimum of three times.