FIGURE 5.

Quantitation of extension from versus excision of a 3′-terminal ribonucleotide by exonuclease-proficient human Pol ϵ. Exonuclease-proficient human Pol ϵ was incubated with all four dNTPs at 25 μm each and 100 nm substrate containing a deoxyribonucleotide primer containing 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 1, 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. The cleavage product resulting from alkaline hydrolysis of an rNMP-containing extension product is indicated (black arrowhead). Products resulting from 3′ excision of the substrate by Pol ϵ are indicated (white arrowhead). All measurements were performed in triplicate with mean values reported.