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 orig in-dependent DNA replication and mutagenesis in a mammalian cell extra ct, double-stranded plasmids containing a single cis,syn-cyclobutane d imer or a pyrimidine-pyrimidone (6-4) photoproduct at a unique TT sequ ence have been constructed. These plasmids have been used as templates in DNA replication-competent extracts from human Beta cells, Plasmids containing a single pyrimidine cyclobutane dimer on the potential lag ging strand for DNA replication are replicated with an efficiency appr oximately equal to that of an unmodified plasmid. A small decrease in replication efficiency of similar to 20% was observed when the lesion was located on the potential leading strand for DNA replication, In bo th orientations, DpnI-resistant, replicated closed circular plasmid DN A was sensitive to nicking by the pyrimidine dimer-specific enzyme, T4 endonuclease V, indicating that complete replication of the damaged p lasmid 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 similar to 50%, indic ating that the mammalian replication complex responds differently to t he two major UV-induced lesions during DNA replication in vitro, Analy sis of the DpnI-resistant, replicated DNA for mutations targeted to th e lesion site indicates that neither of these lesions resulted in sign ificant mutagenesis. UV-induced lesions at TT sites may therefore be p oorly mutagenic under these conditions for DNA replication in human ce ll extracts in vitro.