FIG. 2.

Concentration dependence on the pre-steady-state rate constant of L-3TC-TP incorporation catalyzed by Pol η. A preincubated solution of Pol η (120 nM) and 5′-³²P-labeled 21-41-mer GT DNA (30 nM) (Table 1) was rapidly mixed with increasing concentrations of L-3TC-TP·Mg²⁺ (0.2 μM, •; 0.5 μM, ○; 1 μM, ▪; 2 μM, □; 5 μM, ▴; 10 μM, ▵; 25 μM, ⧫) for various time intervals. (A) A representative gel image is shown for Pol η inserting L-3TC-TP at 25 μM. The lengths of the DNA primer are indicated in the right margin. (B) The concentration of DNA product was plotted as a function of time. The solid lines are the best fits to a single-exponential equation, which determined the observed rate constant, k_obs. (C) The k_obs values were plotted as a function of L-3TC-TP concentration. The data (•) were then fit to a hyperbolic equation, yielding a k_p of 0.0296 ± 0.0009 s⁻¹ and a K_d of 3.3 ± 0.3 μM.