Responses to the Major Acrolein-derived Deoxyguanosine Adduct in Escherichia coli

Abstract

Acrolein, a reactive α,β-unsaturated aldehyde found ubiquitously in the environment and formed endogenously in mammalian cells, reacts with DNA to form an exocyclic DNA adduct, 3H-8-hydroxy-3-(β -D-2′-deoxyribofuranosyl)-5,6,7,8-tetrahydropyrido[3,2-a]purine-9-one (γ-OH-PdG). The cellular processing and mutagenic potential of γ-OH-PdG have been examined, using a site-specific approach in which a single adduct is embedded in double-strand plasmid DNA. Analysis of progeny plasmid reveals that this adduct is excised by nucleotide excision repair. The apparent level of inhibition of DNA synthesis is ∼70% in Escherichia coli ΔrecA, uvrA. The block to DNA synthesis can be overcome partially by recA-dependent recombination repair. Targeted G → T transversions were observed at a frequency of 7 x 10-4/translesion synthesis. Inactivation of polB, dinB, and umuD, C genes coding for "SOS" DNA polymerases did not affect significantly the efficiency or fidelity of translesion synthesis. In vitro primer extension experiments revealed that the Klenow fragment of polymerase I catalyzes error-prone synthesis, preferentially incorporating dAMP and dGMP opposite γ-OH-PdG. We conclude from this study that DNA polymerase III catalyzes translesion synthesis across γ-OH-PdG in an error-free manner. Nucleotide excision repair, recombination repair, and highly accurate translesion synthesis combine to protect E. coli from the potential genotoxicity of this DNA adduct.