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

TLDR

Oxidative DNA damage, notably 8‑hydroxyguanine, is implicated in disease but its in‑vivo base‑pairing specificity remains unclear. The study used two bacteriophage plaque color assays to examine the in‑vivo mutagenic specificity of 8‑hydroxyguanine. The authors employed a reversion assay detecting all three single‑base substitutions from misreading of guanine analogues at a specific site, and a forward mutation assay testing mispairing during incorporation and after multiple incorporations into an M13mp2 single‑stranded gap. oh8Gua induced a 0.7 % mutation frequency with 22 of 23 G→T substitutions in the reversion assay, and 16 % mutants in the forward assay, predominantly A→C (76/78) with two G→T, showing that as template it causes G→T and as substrate it causes A→C substitutions via mispairing.

Abstract

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 mutagenic 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.