8-hydroxyguanine, an abundant form of oxidative DNA damage, causes G → T and A → C substitutions

Mutations caused by oxidative DNA damage may contribute to human disease. A major product of that damage is 8-hydroxyguanine (oh⁸Gua). Because of differences in experimental design, the base pairing specificity of oh⁸G in vivo is not completely resolved. Here, oh⁸dGTP and DNA polymerase were used in two complementary bacteriophage plaque color assays to examine the mutagenic specificity of oh⁸Gua in vivo. The first is a reversion assay that detects all three single-base substitutions caused by misreading of guanine analogues inserted at a specific site. oh⁸Gua at that site gave a mutation frequency of 0.7%. Twenty-two of the 23 mutations were G → T substitutions. The second assay, a forward mutation assay, tests the mispairing potential of any altered nucleotide 1) during incorporation as substrate nucleotide, and 2) after multiple incorporations into a single-stranded DNA gap region of M13mp2. Substituting oh⁸dGTP for dGTP during polymerization produced 16% mutants; two classes of mutations were observed, both caused by pairing of oh⁸Gua with A. Seventy-six of 78 mutations were A → C substitutions, and two were G → T substitutions. These assays thus illustrate mutagenie replication of oh⁸Gua as template causing G → T substitutions and misincorporation of oh⁸Gua as substrate causing A → C substitutions, both caused by oh⁸Gua · A mispairs.