Figure 1.

Incorporation of Rh·dTTP opposite dA at varying concentrations of MgCl₂. The final reaction conditions for all experiments depicted consisted of 100 nM RB69 pol, 90 nM DNA, 250 μM Rh·dTTP, 50 mM MOPS (pH 7.0), and varying concentrations of MgCl₂ from 0.5 to 100 mM. (A) The reaction was initiated by premixing Rh·dTTP and MgCl₂ and then rapidly mixing this solution with one containing RB69 pol and DNA. The incorporation rate is 92 ± 15 s⁻¹. (B) The reaction was initiated by premixing RB69 pol, DNA, and MgCl₂ and then rapidly mixing this solution with one containing Rh·dTTP. The incorporation rate is 77 ± 6 s⁻¹. (C) The reaction was initiated by premixing RB69 pol, DNA, and MgCl₂ and then rapidly mixing this solution with one containing Rh·dTTP and MgCl₂. The incorporation rate is 80 ± 10 s⁻¹. (D) The rates of product formation are plotted vs MgCl₂ concentration for each of the three experiments depicted in panels A–C. Regardless of when MgCl₂ is added, the observed maximum rate of the reaction fits to approximately 80 s⁻¹, at a MgCl₂ concentration of approximately 10 mM. Because of the mixing procedure, each solution was prepared at twice the final reported concentration. The product concentrations were plotted vs time and fit to a single-exponential equation.