GENETIC-EFFECTS OF OXIDATIVE DNA DAMAGE - COMPARATIVE MUTAGENESIS OF 7,8-DIHYDRO-8-OXOGUANINE AND 7,8-DIHYDRO-8-OXOADENINE IN ESCHERICHIA-COLI

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZalpha gene of bacteriophage M13 DNA induces a targeted G --> T transversion after replication in Escherichia coli (Biochemistry, 29, 7024 - 7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO . base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A-8OXOG)] was synthesized. After 5'-phosphorylation with [gamma-P-32] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into Ecoli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistinguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.