Abstract

The interaction of 5-trifluoromethyl-2’-deoxyuridine 5’-triphosphate (F₃dThd-5'-PPP) with various DNA-directed DNA polymerases has been studied. This analogue exhibited competitive inhibition with dThd-5'-PPP for partially purified DNA polymerases from vaccinia virus-infected HeLa cells, uninfected HeLa cell cytoplasm and nuclei, and calf thymus. The inhibition produce by F₃dThd-5'-PPP of the incorporation of deoxycytidine 5'-triphosphate and deoxyadenosine 5'-triphosphate was noncompetitive or mixed. The K_m values for dThd-5'-PPP of vaccinia virus-induced, HeLa cell cytoplasmic, HeLa cell nuclear, and calf thynmus DNA polymerases were 3.4, 4.6, 5.1, and 10 µM, respectively. The corresponding K_i values of F₃dThd-5'-PPP for these enzymes were 4.0, 8.5, 11.5, and 17 µM. F₃dThd-5'-PPP was incorporated into DNA by all these DNA polymerases. This incorporation was inhibited competitively by dThd-5'-PPP. The rates of F₃dThd-5'-PPP incorporation by these enzymes were slower than those of dThd-5'-PPP. The ratio of the incorporation rate of the analogue to that of dThd-5'-PPP was higher with vaccinia virus polymerase than with uninfected HeLa cell enzymes.