Figure 4.

Effects of GA-FAPy-dfG on DNA polymerases activity.A, primer extension assay past GA-FAPy-dfG by Polε. A [³²P]-labeled 15-mer primer was annealed with 30-mer template oligonucleotides carrying dG (lanes 1 and 4), dfG (lanes 2 and 5), or GA-FAPy-dfG (lanes 3 and 6) at the 16th nucleotide from the 3′ terminus (denoted as X). The primer–template substrates were then incubated with (lanes 4–6) or without (lanes 1–3) Polε for 15 min at 37 °C. The products were subjected to polyacrylamide gel electrophoresis under denaturing conditions and then autoradiographed. B, primer extension assay past GA-FAPy-dfG by Polη, Polκ, Polι, Polβ, and REV1. A [³²P]-labeled 15-mer primer was annealed with 30-mer template oligonucleotides carrying dG (lanes 1, 4, 7, 10, 13, and 16), dfG (lanes 2, 5, 8, 11, 14, and 17), or GA-FAPy-dfG (lanes 3, 6, 9, 12, 15, and 18) at the 16th nucleotide from the 3′ terminus (denoted as X). The primer–template substrates were then incubated with Polη (lanes 4–6), Polκ (lanes 7–9), Polι (lanes 10–12), Polβ (lanes 13–15), REV1 (lanes 16–18), or without enzymes (lanes 1–3) for 15 min at 37 °C. C, primer extension assay by Polζ using 16-mer primers, 16C and 16T, which placed the 3′ dC or dT of the primer opposite dG, dfG, or GA-FAPy-dfG. The [³²P]-labeled primers (15, lanes 1–6; 16C, lanes 7–12; 16T, lanes 13–18) were annealed with 30-mer template oligonucleotides carrying dG (lanes 1, 4, 7, 10, 13, and 16), dfG (lanes 2, 5, 8, 11, 14, and 17), or GA-FAPy-dfG (lanes 3, 6, 9, 12, 15, and 18) at the 16th nucleotide from the 3′ terminus (denoted as X). The primer–template substrates were then incubated with (lanes 4–6, 10–12, and 16–18) or without (lanes 1–3, 7–9, and 13–15) Polζ for 15 min at 37 °C. The products were subjected to polyacrylamide gel electrophoresis under denaturing conditions and then autoradiographed. GA-FAPy-dfG, N⁶-(2-deoxy-2-fluoro-d-arabinofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine.