MAMMALIAN DNA POLYMERASE-ALPHA, POLYMERASE-BETA, POLYMERASE-GAMMA, POLYMERASE-DELTA, AND POLYMERASE-EPSILON INCORPORATE FIALURIDINE (FIAU) MONOPHOSPHATE INTO DNA AND ARE INHIBITED COMPETITIVELY BY FIAU TRIPHOSPHATE

Fialuridine [FLAU, -2'-fluoro-beta-D-arabinofuranosyl)-5-iodouridine] was used in clinical trials for chronic hepatitis B virus infection an d was extremely toxic. Evidence suggested targets of FIAU toxicity inc luded mitochondria, but toxic mechanisms were unclear. Since FLAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) co uld be incorporated into mitochondrial DNA by DNA pol-gamma and into g enomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All fiv e purified mammalian DNA polymerases incorporated FIAUMP into the nasc ent DNA chain during in vitro DNA synthesis. When FIAUTP was substitut ed for dTTP, oligonucleotide products were generated efficiently by DN A pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were signi ficantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited t he accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The K- i with DNA pol-gamma was 0.04 mu M, the lowest K-i among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FLAU toxicity.