Effect of 2′, 3′-dideoxythymidine-5′-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase α is the only polymerase required for cellular DNA replication

Abstract

We have studied the effects of the nucleotide analogue, 2′,3′-dideoxythymidine-5′-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100–500 μM when the dTTP concentration is maintained at 10 μM. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases β or γ is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase α is very resistant. These observations suggest that DNA polymerase α may be the only DNA polymerase required for all aspects of cellular DNA synthesis.