Studies on direct and indirect effects of DNA damage on mutagenesis in monkey cells using an SV40-based shuttle vector.
We are using an SV40-based shuttle vector, pZ189, to study mechanisms of mutagenesis in mammalian cells. The vector can be treated with mutagens in vitro and replicated in animal cells; resulting mutants can be selected and amplified in bacteria for DNA sequencing. This versatile vector system has allowed us to explore several different questions relating to the mutagenic process. We have studied the direct effects of template damage caused by UV or benzo[a]pyrene diolepoxide by treating vector DNA with these agents and then replicating the damaged DNA in monkey cells. Mutational mechanisms were deduced from the spectrum of mutations induced in the supF target gene of the vector DNA. To study the role of indirect effects of DNA damage on mutagenesis in mammalian cells, we have treated the cells and the vector DNA separately with DNA-damaging agents. We find that pretreatment of cells with DNA-damaging agents, or with conditioned medium from damaged cells, causes an enhancement of mutagenesis of a UV-damaged vector. Thus, DNA damage can act indirectly to enhance the mutagenic process. We also have preliminary evidence that pZ189 can be used in an in vitro DNA replication system to study the process of mutation fixation on the biochemical level. We believe that the pZ189 vector will prove to be as useful for in vitro studies of mutational mechanisms as it has been for in vivo studies.
Dixon
K
K
Section on Viruses and Cellular Biology, National Institute of Child Health and Human Development, Bethesda, MD 20892.
Roilides
E
E
Hauser
J
J
Levine
A S
AS
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004249:DNA Damage; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D000882:Haplorhini; D016685:Mitomycin; D008937:Mitomycins / Q000494:pharmacology; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests / Q000379:methods*; D012365:RNA, Transfer, Tyr; D013539:Simian virus 40 / Q000235:genetics*; D013489:Suppression, Genetic; D014466:Ultraviolet Rays
D008937:Mitomycins; D012365:RNA, Transfer, Tyr; D016685:Mitomycin; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide