SITE-SPECIFIC MUTAGENESIS INDUCED BY SINGLE O-6-ALKYLGUANINES (O-6-N-PROPYL, O-6-N-BUTYL, O-6-N-OCTYL) IN-VIVO

The mutagenic activity of a series of longer chain O6-n-alkylguanine r esidues (O6-n-propyl, O6-n-butyl, O6-n-octyl) has been analyzed using a plasmid molecule (pUC 9) in which single O6-alkylguanines were posit ioned in the unique Pstl recognition site by shot gun ligation (Nuclei c Acids Res. 13, 3305 - 3316 (1985)) of overlapping synthetic oligonuc leotides. After transfection of these vectors into E.coli cells having normal DNA repair systems, progeny plasmids were produced, of which 2 .6%, 2.8% and 4.3% were mutated in their Pstl site when containing O6- n-propylguanine, O6-n-butylguanine, O6-n-octylguanine, respectively. D NA sequence analysis of mutant plasmid genomes revealed that O6-n-prop ylguanine and O6-n-butylguanine induced exclusively G --> A transition s located specifically at the preselected site. O6-n-octylguanine indu ced apart from G --> A transitions (70%) also targeted G --> T transve rsions (30%). These results indicate that the mutation frequency of lo nger chain O6-alkylguanines can be substantial in cells with normal re pair systems and that the mutation pattern depends on the nature of th e alkyl group.