FIGURE 4.

Extension from a 3′-terminal ribonucleotide by exonuclease-deficient human Pol ϵ. Exo− human Pol ϵ was incubated with all four dNTPs at 25 μm each and a substrate containing a deoxyribonucleotide primer with a single 3′-terminal rGMP (left panel) or rUMP (right panel) nucleotide. 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 (−Pol ϵ). Products were untreated (−) or incubated at 55 °C for 2 h in either KCl (Cl) or KOH (OH). Products were resolved on a 12% denaturing acrylamide gel.