ANALYSIS OF MUTATIONS INDUCED BY REPLICATION OF UV-DAMAGED PLASMID DNA IN HELA-CELL EXTRACTS

We have used an SV40-based shuttle vector, pZ189, to investigate the c apacity of Hela cell extracts to reproduce the in vivo process of muta tion fixation. We showed previously that when UV-irradiated pZ189 is r eplicated in these extracts, bypass of UV photoproducts occurs, result ing in bose substitution mutations in the supF gene of the vector. Her e we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single o r tandem double base substitutions at dipyrimidine sites; of these, ap proximately 90% ore G:C-->A:T transitions. Mutations are observed pred ominantly at a few sites, in particular at positions 155 and 156 in th e supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with resp ect to the SV40 origin of replication, suggesting that mutation fixati on occurs similarly on both the leading and the lagging strands for DN A replication. Generally, the mutational hot spots observed in vitro o re at the same sites as those observed when UV-irradiated pZ189 was pa ssaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in Hela cell extra cts accurately reflects these processes in the intact cell. (C) 1995 W iley-Liss, Inc.