Figure 5.

Steady-state kinetic analysis of dATP insertion by hpol η during translesion synthesis. PAGE (20%, 7 m urea) of steady-state kinetic analysis using hpol η (1–2.3 nm) with varying concentrations of dATP (0–800 μm, indicated with wedges) was conducted for DNA/DNA-dA (A), DNA/DNA-rA (B), and DNA/DNA-1,N⁶-ϵrA (C). All reactions were carried out at 37 °C for 5 min in duplicate. The indicated data points are shown as means ± S.D. (error bars); see Table 1 for k_cat and K_m values (estimated using fits to a hyperbolic equation in Prism (GraphPad)); see “Experimental procedures” and Table S1 for the oligonucleotide sequences. P, FAM-labeled DNA primer.