Genetic effects of oxidative DNA damage— comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro8-oxoadenine in E.coli

A single 7,8-dihydro-8-oxoguanine (G8^3^; 8-hydroxy-guanine) adduct in the lacZa 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 Q8-oxo.base pair, where G8<)XO is in the syn conforma-tion about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-°xo; 8-hydroxy-adenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-°x0, as a mono-nucleoside A8*0*0 assumes the syn conformation. This work has assessed the relative mutagenicities of A8-oxo a n ( j Q8-OXO;n the same experimental system. A deoxypentanucleotide containing A8 O X O [d(GCT-A8-0X0G)] was synthesized. After 5'-phosphorylation with [7-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique Nhe\ restriction site. Genomes containing either A 8 " 0 * 0 (at position 6275, [ +] strand) or G8-°xo (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-°xo induced G—T transversions at an apparent frequency of-0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8^>xo-modified DNA were almost indistinguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-°xo is at least an order of magnitude less mutagenic than G8'0'50 in E.coli cells with normal DNA repair capabilities