The in vivo mutagenic frequency and specificity of O6-methylguanine in phi X174 replicative form DNA.
A bacteriophage phi X174-based site-specific mutagenesis system for the study of the in vivo mutagenic frequency and specificity of carcinogen-induced modification in DNA is presented. A (-)-strand primer containing O6-methylguanine in a specific site was hybridized to a single-stranded region in gene G of phi X gapped duplex DNA. The hybrid was enzymatically converted to replicative form DNA and was used to transform Escherichia coli cells. All gene G mutants generated by the modification were rescued by genetic complementation. An amber mutation in lysis gene E of the (+) strand of the replicative form DNA prevented lytic growth of wild-type phage derived from this strand. In each mutant-containing infective center produced from the transformed cells, gene G mutant phage were present in a 3:1 ratio compared to wild type. Thus, in vivo, O6-methylguanine in replicating phi X DNA has a mutagenic frequency of 75%. When repair of O6 methylguanine occurred, it was prereplicative. The mutations were due exclusively to the misincorporation of thymine.
D010584:Bacteriophage phi X 174 / Q000235:genetics*; D001483:Base Sequence; D004260:DNA Repair; D004261:DNA Replication; D005814:Genes, Viral; D006147:Guanine / Q000031:analogs & derivatives* / Q000502:physiology; D006860:Hydrogen Bonding; D009154:Mutation
D006147:Guanine; C008449:O-(6)-methylguanine