Mechanism of ultraviolet-induced mutagenesis: Extent and fidelity of in vitro DNA synthesis on irradiated templates

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Abstract
The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded .vphi.X174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol+ and polA1 mutants and purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on UV-irradiated .vphi.X174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated .vphi.X174 DNA was observed. This nucleotide turnover is apparently due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3'' .fwdarw. 5'' proofreading exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase .alpha., both of which are devoid of any exonuclease activity, was only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. UV mutagenesis in bacteria probably requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3'' .fwdarw. 5'' exonuclease activity associated with DNA polymerases.