Site-Specific Mutagenesis in Escherichia coli by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives in Double-Stranded or Gapped Plasmids.

ABSTRACT

PURPOSE: 7-Bromomethylbenz[alpha]anthracene is a known mutagen and carcinogen. The mutagenic potency of its two major DNA adducts, i.e., N2-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[alpha]a2G) and N6-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyadenosine (b[alpha]a6A), as well as the simpler benzylated analogs, N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A), were determined in E. coli. MATERIALS AND METHODS: Double-stranded and gapped plasmid vectors were used to determine the mutagenicity of b[alpha]a2G, b[alpha]a6A, bn2G and bn6A in E. coli. The four, suitably protected, bulky exocyclic amino-substituted adducts were incorporated into 16-base oligodeoxyribonucleotides, in place of normal guanine or adenine residues, which form part of the ATG initiation codon for the lacZ' alpha-complementation gene. The site-specifically modified oligodeoxyribonucleotides were then incorporated into double-stranded plasmids, which contained uracil residues in the complementary strand in the vicinity of the initiation codon. The uracil residues lead to the creation of a gap in the complementary strand due to the actions of E. coli uracil-DNA glycosylase and AP endonuclease. Following the transfection of these plasmid vectors into E. coli strain GP102, a lacZ alpha complementing version of the parent strain AB1157, their propensity to induce mutation was investigated. RESULTS: The percentages of mutant colonies produced by the four modified nucleosides, in both the double-stranded and gapped plasmid vectors, were not significantly different from those produced by the unmodified plasmids. The mutagenicities of the b[alpha]a2G and b[alpha]a6A were extremely low, and a totally unexpected result, whereas, those of the bn2G and bn6A were undetectable. CONCLUSION: In this E. coli site-specific mutagenesis system, these bulky aralkylated adducts exhibited no significant mutagenicities, either with or without SOS induction.