Kinetics of correct dCTP incorporation onto 20-mer/26-mer-dGAP catalyzed by hPolη under single-turnover conditions. (A) A preincubated solution of hPolη (130 nM) and 5′-[32P]-labeled 20-mer/26-mer-dGAP (20 nM) was rapidly mixed with increasing concentrations of dCTP (25 μM, •; 50 μM, ■; 100 μM, △; 200 μM, ▲; 400 μM, ○; 800 μM, ◆; 1200 μM, □) for various time intervals at 37 °C. Each time course was fit to Equation 4 to obtain a kobs for a specific dCTP concentration. (B) The plot of kobs values against dCTP concentrations was fit to Equation 5 to yield a Kd, dCTP of 287 ± 24 μM and a kp of 8.1 ± 0.2 s−1.