FIG. 8.

(S)-HPMPA inhibits labeling with TdT. (A) Primer P11 was annealed to strand T17 and incubated at 37°C with vaccinia DNA polymerase (1 min) or MMLV reverse transcriptase (1 h), in the presence of 10 μM dGTP and either 10 μM dATP or 10 μM (S)-HPMPApp. The T17 strand was degraded using UDG and heat, and the biotinylated P11 strand was recovered by using magnetic beads. The DNAs were labeled using terminal transferase and [α³²P]3′- deoxyATP and subjected to electrophoresis, and the radioactivity was detected by using a phosphorimager. Primer P11 was purified and labeled the same way but not incubated with either polymerase (lane 1). (B) Primer P20 was 5′ end labeled and annealed to template T17 and then incubated with vaccinia polymerase or MMLV reverse transcriptase as described above. The reaction products were size fractionated by electrophoresis and detected by using a phosphorimaging. Both polymerases can incorporate (S)-HPMPA (and dGMP) into DNA, as judged by using prelabeled primers (lanes 8 and 10), but these N+1 products are not postlabeled with terminal transferase (lanes 3 and 5). The TdT is still active, as shown by the capacity to label any of the molecules encoding dAMP (lanes 2 and 4). VAC, vaccinia DNA polymerase; RT, reverse transcriptase.