Analysis of mutations induced by replication of UV-damaged plasmid DNA in HeLa cell extracts
- Univ. of Cincinnati College of Medicine, OH (United States); and others
We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C{r_arrow}A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell. 37 refs., 4 figs., 3 tabs.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 258495
- Journal Information:
- Environmental and Molecular Mutagenesis, Journal Name: Environmental and Molecular Mutagenesis Journal Issue: 2 Vol. 26; ISSN 0893-6692; ISSN EMMUEG
- Country of Publication:
- United States
- Language:
- English
Similar Records
Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F
Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells