Complete replication of plasmid DNA containing a single UV-induced lesion in human cell extracts

To investigate the effect of the major UV-induced lesions on SV40 origin-dependent DNA replication and mutagenesis in a mammalian cell extract, double-stranded plasmids containing a single cis,syn-cyclobutane dimer or a pyrimidine-pyrimidone (6-4) photoproduct at a unique TT sequence have been constructed. These plasmids have been used as templates in DNA replication-competent extracts from human HeLa cells. Plasmids containing a single pyrimidine cyclobutane dimer on the potential lagging strand for DNA replication are replicated with an efficiency approximately equal to that of an unmodified plasmid. A small decrease in replication efficiency of approximately 20% was observed when the lesion was located on the potential leading strand for DNA replication. In both orientations, DpnI-resistant, replicated closed circular plasmid DNA was sensitive to nicking by the pyrimidine dimer-specific enzyme, T4 endonuclease V, indicating that complete replication of the damaged plasmid occurs in vitro. In contrast, a (6-4) photoproduct, within the same site and sequence context on the lagging strand for DNA synthesis, inhibits replication in vitro by an average of approximately 50%, indicating that the mammalian replication complex responds differently to the two major UV-induced lesions during DNA replication in vitro. Analysis of the DpnI-resistant, replicated DNA for mutations targeted to the lesion site indicates that neither of these lesions resulted in significant mutagenesis. UV-induced lesions at TT sites may therefore be poorly mutagenic under these conditions for DNA replication in human cell extracts in vitro.