Mutagenesis and repair of O6-substituted guanines

The mutagenic activity of O6-methylguanine has been investigated using a single-stranded M13mp8 phage DNA molecule in which a single O6-methylguanine residue was positioned in the unique recognition site for the restriction endonuclease, Pst I. After introduction of this vector into Escherichia coli, progeny phage were produced, of which 0.4% were mutated in their Pst I site. To determine the impact of DNA repair on mutagenesis, levels of O6-methylguanine-DNA methyltransferase (an O6-methylguanine repair protein) were depleted in host cells by treatment with N-methyl-N'-nitro-N-nitrosoguanidine prior to viral DNA uptake. In these cells, the mutation frequency due to O6-methylguanine increased with increasing N-methyl-N'-nitro-N-nitrosoguanidine dose (the highest mutation frequency observed was 20%). DNA sequence analysis of mutant genomes revealed that O6-methylguanine induced G to A transitions, exclusively.