The Oxidative DNA Lesion 6-Oxo-M1dG is a Potent Replication Block, Inducing Deletions and Base Substitution Mutations In Vivo

Abstract

One of the most prevalent exocyclic DNA adducts is 3-(2-deoxy-β-D-erythro-pentofuranosyl) pyrimido[1,2-α]purin-10(3H)-one (M1dG), an oxidative DNA lesion that forms by the reaction of guanines in nucleic acids and pool nucleotides with oxidation-induced base propenals or with the lipid peroxidation product, malondialdehyde. Further oxidation converts M1dG to 6-oxo-M1dG, an even more deleterious lesion whose genotoxic and mutagenic properties have been characterized in vitro. The present work uses a site-specifically modified viral genome to evaluate the biochemical consequences of 6-oxo-M1dG in Escherichia coli cells and contrast them with the properties of the M1dG parent lesion. We found that 6-oxo-M1dG strongly inhibited replication, with a bypass efficiency of 1–2%, relative to an unmodified guanine. By contrast, under the same experimental conditions, the bypass efficiency of M1dG was 30–40%. Beyond its low bypass rate, 6-oxo-M1dG was 20 times more mutagenic than M1dG, with the majority of mutations being single base deletions. However, when the levels of bypass polymerases were increased by inducing the SOS response, the proportion of deletions decreased, at the expense of additional single base substitutions, primarily G → T and G → C mutations. Finally, two DNA repair pathways─the direct reversal dioxygenase AlkB and glycosylase MutY─were investigated for their putative activity on 6-oxo-M1dG. The results indicated that neither system was capable of repairing this highly mutagenic lesion.