FIG. 5.

Vaccinia DNA polymerase can excise (S)-HPMPA from the primer terminus but not if (S)-HPMPA is the penultimate 3′-nucleotide. Primer P1 was labeled with ³²P, annealed to template T11, and incubated for 1 min with 2.5 ng of vaccinia DNA polymerase/μl at 25°C in the presence of 10 μM dATP, 10 μM (S)-HPMPA, 10 μM (each) dATP and dTTP or 10 μM (each) (S)-HPMPA and dTTP. This produced molecules bearing the 3′ structures indicated in the Figure [“H” = (S)-HPMPA]. The unincorporated nucleotides and (S)-HPMPApp were removed by gel filtration, and the purified substrates were incubated with fresh enzyme at 25°C. The reactions were sampled at the times indicated, mixed with formamide stop buffer, and size fractionated on 10% polyacrylamide gels, and the radioactivity was detected by phosphorimaging. Water was substituted for DNA polymerase in the “no polymerase” controls (indicated by “−” symbols). Primers terminated with (S)-HPMPA+dTMP are highly resistant to exonuclease attack (lanes 14 to 18, at right) but did not inhibit exonuclease attack on a trace of contaminating molecules terminated with dAMP or (S)-HPMPA (asterisk).