Figure 3.

Fidelity of Pol η opposite the first damaged template T of the cis-syn T–T dimer. (A) Deoxynucleotide incorporation across from the first template T of the T–T dimer (substrate S-3; Fig. 1). Pol η (1 nM) was incubated for 5 min at 25°C with the primer-template DNA (10 nM) and with increasing concentrations of the incorrect deoxynucleotide (dGTP, dTTP, and dCTP; 0–500 μM) or the correct deoxynucleotide (dATP; 0–20 μM). The reactions were stopped and examined by denaturing PAGE. A portion of the template sequence is shown on the left. The asterisk indicates the ³²P-labeled 5′ end of the primer. T–T dimer. (B) Quantitation of the deoxynucleotide incorporation reactions. The observed rate of deoxynucleotide incorporation is plotted as a function of concentration for each of the deoxynucleotides. The data were fit by using Eq. 1, and the V_max and K_m parameters obtained from the fit are listed in Table 1.