Figure 3.
Steady-state kinetic analysis of individual dATP and dCTP insertions by hpol η. Reactions contained templates dG (A and D), 2′-F dG (B and E), and N7-CH3 2′-F dG (C and F) at position X in the sequences 5′-CGGGCTCGTAAGCGTCAT-3′ and 3′-GCCCGAGCATTCGCAGTAXTACT-5′. Reactions were done at 37 °C for 5–10 min by incubating 120 nm primer–template oligonucleotide complex. For different panels, different hpol η concentrations were used as indicated. For A, B, C, and F, 5 nmol of enzyme was used, and the reaction was done for 5 min, with varying concentrations of dATP and dCTP. In the D and E, 2.5 nmol of enzyme was used, and the reaction was conducted for 5 min, with varying concentrations of dATP and dCTP. Fitting was to a hyperbolic equation in GraphPad Prism version 8.0, and kcat and Km values are presented in Table 1.