Effects of DNA Polymerase Inhibitors on Replicative and Repair DNA Synthesis in Ultraviolet-irradiated HeLa Cells

Abstract

Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2′,3′-dideoxythymidine 5′-triphosphate (d₂TTP) inhibits DNA polymerase β and γ but not α. 1-β-D-Arabinofuranosylcytosine 5′-triphosphate (araCTP) inhibits both DNA polymerase α and β although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-β-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2′,3′-dideoxythimidine (d₂Thd, a precursor of d₂TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d₂Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d₂TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d₂TTP effectively inhibited repair synthesis. The microinjection of d₂TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis.