Fig. 5.

(A) Autoradiograms of a TLC analysis of the products from a [γ−³²P]ATP phosphate transfer assays with UMP-CMPK (1–4) or TMPK (6–9) using dCMP (1, 6), dTMP (2, 7), N5-MP (3, 8), and N5-2OH-MP (4, 9) as substrates. Lane 5, [γ−³²P]ATP only. (B) Autoradiogram of a TLC analysis of the diphosphate products from a [γ−³²P]ATP phosphate transfer assay with NDPK using dTDP (2, 3) and N5-DP (4, 5) as substrates. The assays were performed for 30 (2, 4) and 60 (3, 5) minutes. [γ−³²P]ATP (1) served as a control. (C and D) Autoradiograms of a gel analysis of the reaction products of dTTP, AZT-TP, and N5-TP with Klenow DNA polymerase I using a running start primer-template DNA. (C 1–6) Incorporation of dTTP into the running start primer at six different concentrations of dTTP (0, 1, 10, 25, 50, and 100 μM). (C 7–9) Incorporation of the dTTP product from a coupled synthesis using dThd and TK1, TMPK, and NDPK, as described in Materials and Methods. (Dilutions: C7 = 1/16, C8 = 1/8, C9 = 1/4.) The 16-mer primer template was incubated with T4 polynucleotide kinase and [γ−³²P]ATP for 10 minutes at 37°C leading to formation of a ³²Pi-labeled 18-mer nucleotide product (C1 and D1). Additions of dTTP (C 1–6) or dTTP (D 2–5), AZT-TP (D 6–9), and N5-TP (D 10, 11) from coupled reactions and Klenow DNA polymerase to the labeled 18-mer only leads to the formation of ³²Pi-labeled 19-mer nucleotide products for dTTP and AZT-TP (Dilutions: D2/D6 = 0, D3/D7 = 1/2, D4/D8 = 1/4, D5/D9 = 1/8, D10 = 0, D = 1/2). Reactions were terminated by addition of 2 volumes of 90% formamide, 0.25 M EDTA. Products were analyzed on 15% polyacrylamide-7 M urea gels and detected by phosphorimaging (1 hour).