FIGURE 6.

Pol ϵ extension from increasing numbers of 3′-terminal ribonucleotides. Human Pol ϵ was incubated with substrates containing primer oligonucleotides with increasing numbers of 3′-terminal ribonucleotides (Table 2). Primers have sequences identical to the fully DNA primers described above (R0) but with one, two, three, four, or five consecutive ribonucleotides at the 3′-end (R1–R5). Reactions were carried out at 37 °C for the indicated times using 25 μm dNTPs, 100 nm substrate, and either 1 nm Exo− (A) or Exo+ Pol ϵ (B). Unreacted substrates (− hPol ϵ) were either untreated or treated with KOH to demonstrate alkaline hydrolysis of the ribonucleotide-containing substrates. Aliquots of Pol ϵ extension products were taken at the indicated times (+ hPol ϵ) and treated with either salt (+ KCl) or alkali (+ KOH). All measurements were performed in triplicate with mean values reported. ′, minutes.