FIGURE 3.

Bypass of template rU by human Pol ϵ. Human Pol ϵ was incubated with all four dNTPs at 25 μm each and 100 nm substrate containing either entirely template deoxyribonucleotides (undamaged) or a single rUMP at the N + 2 position (rU) where N is the unreacted primer. Reactions were carried out as described under “Experimental Procedures.” Reactions were performed at 37 °C and started with the addition of 1 nm enzyme, and aliquots were removed at 2, 5, and 10 min (′). Control substrate with no enzyme added is shown (−). Products were resolved on a 12% denaturing acrylamide gel. Bypass probabilities, bypass efficiency, and termination probabilities were calculated as described (see “Experimental Procedures” and Refs. 31 and 32) using the following equations: Bypass probability = ΣBand intensity_{(>N + 2)}/ΣBand intensity_(>N), Bypass efficiency = (Bypass probability_rU)/(Bypass probability_undamaged), and Termination probability = Band intensity_(n)/ΣBand intensity_(≥n).