FIG. 10.

Effects of templates bearing nucleoside phosphonate drugs on trans-lesion DNA synthesis. Templates were prepared bearing dCMP/CDV (upper panels) or dAMP/(S)-HPMPA (lower panels) and annealed to the six indicated ³²P-labeled primer strands. These primers terminate at positions N+1 (P10 and P17), N (P13 and P18), and N−1 (P14 and P19), where N is the site of drug incorporation [“X” = CDV, “H” = (S)-HPMPA]. Each of these primer-template pairs was incubated with 2.5 ng of vaccinia DNA polymerase/μl at 37°C for 0, 1, 2, 5, 10, or 15 min (triangles) in the presence of all four dNTPs (50 μM each), and the products were recovered by using magnetic beads. The reaction products were then separated on a 10% polyacrylamide gel, and the radioactivity was visualized by phosphorimaging. All of the six primers were rapidly extended across control molecules bearing dCMP or dAMP residues. In contrast, P10 and P17 were extended only one nucleotide (left-hand column), and the drugs blocked net DNA synthesis from the other four primers. Each of the enzymatically prepared template strands was also separately labeled with terminal transferase and [α³²P]3′-deoxyATP to measure the length of the original extension products (“T”). The electrophoretic properties of unmodified primer strands are illustrated in lanes marker “P.”