Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells

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Abstract
We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagencity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T .fwdarw. C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T .fwdarw. A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than than expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.