Mammalian DNA polymerases alpha, beta, gamma, delta, and epsilon incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU Triphosphate.
Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA 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 significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki 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 FIAU toxicity.
Lewis
W
W
Department of Pathology and Laboratory Medicine, College of Medicine, University of Cincinnati, Ohio 45267-0529.
Meyer
R R
RR
Simpson
J F
JF
Colacino
J M
JM
Perrino
F W
FW
D000818:Animals; D001086:Arabinofuranosyluracil / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D004247:DNA / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D008928:Mitochondria / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors; D013379:Substrate Specificity; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
C090682:5-iodo-2'-fluoroarabinosyluridine triphosphate; D003856:Deoxyuracil Nucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides; D001086:Arabinofuranosyluracil; C043457:fialuridine; D004247:DNA; C090779:fialuridine monophosphate; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate