Figure 3.

Optimal divalent cation conditions and polymerization rates for ϕ6 wt and E491Q RdRps. (A) Manganese dependence of the wt and E491Q polymerases in replication reactions with 5 mM MgCl₂ and without magnesium. Reaction mixtures containing increasing concentrations of MnCl₂, 50 μg/ml sΔ⁺₁₃ ssRNA template and 270 nM polymerase were incubated at 30°C for 2 h. The reactions were analysed by standard gel electrophoresis and quantified with a phosphoimager (Fuji BAS1500). (A) The graph is normalized setting the highest observed value within each plot to 1. (Right Panel) A phosphorimager analysis of the reactions with 0–2 mM MnCl₂ and without MgCl₂. The graph is normalised setting the value obtained with 5 mM MnCl₂ to 1 within each plot. (B) Time-dependent accumulation of wt- and E491Q-directed replication products. Standard reaction mixtures containing 2 mM MnCl₂, 5 mM MgCl₂, 270 nM polymerase and 110 μg/ml l⁺ ssRNA template were incubated at 30°C and 10 μl aliquots, sampled at the time points indicated, were analysed on a standard agarose gel. Inserts show the original gels used for the phosphoimager quantification.