FIGURE 3.
Pre-steady-state burst kinetics of incorporation opposite G, N2-BzG, N2-NaphG, N2-AnthG, N2-BPG, O6-MeG, O6-BzG, and O6-PobG by human REV1. REV1 (120 nm) was incubated with 100 nm 24-mer/36-mer primer-template complex in the rapid quenched-flow instrument and mixed with 1 mm dCTP (and MgCl2) to initiate reactions. A, G and N2-G adducts: 24-mer/36-G-mer (▪), 24-mer/36-N2-BzG-mer (▴), 24-mer/36-N2-NaphG-mer (▾), 24-mer/36-N2-AnthG-mer (♦), 24-mer/36-N2-BPG-mer (•). B, G and O6-G adducts: 24-mer/36-G-mer (▪), 24-mer/36-O6-MeG-mer (▴), 24-mer/36-O6-BzG-mer (▾), and 24-mer/36-O6-PobG-mer (♦). All polymerization reactions were quenched with 0.3 m EDTA at various time intervals. The data were fit to the burst equation y = A(1 – e–kpt) + ksst, as described under “Experimental Procedures” (without normalization of kss for enzyme concentration in the equation). The following rates were estimated: G-mer, kp = 0.43 ± 0.13 s–1, kss = 0.015 ± 0.001 s–1; N2-BzG-mer, kp = 0.62 ± 0.21 s–1, kss = 0.014 ± 0.001 s–1; N2-NaphG-mer, kp = 0.22 ± 0.07 s–1, kss = 0.0045 ± 0.0008 s–1; N2-AnthG-mer, kp = 0.26 ± 0.09 s–1, kss = 0.0033 ± 0.0008 s–1; N2-BPG-mer, kp = 0.88 ± 0.22 s–1, kss = 0.0028 ± 0.0006 s–1; O6-MeG-mer, kp = 0.08 ± 0.03 s–1, kss = 0.0037 ± 0.0007 s–1; O6-BzG-mer, kp = 0.15 ± 0.05 s–1, kss = 0.0056 ± 0.0004 s–1.