Mutagenicity in Escherichia coli of the Major DNA Adduct Derived from the Endogenous Mutagen Malondialdehyde

• Published in: Proceedings of the National Academy of Sciences - PNAS 1997-08, Vol.94 (16), p.8652-8657
• Main Authors: Fink, Stephen P., Reddy, G. Ramachandra, Marnett, Lawrence J.
• Format: Article
• Language: English
• Subjects: Adducts; Bacteria; Biological Sciences; Deoxyribonucleic acid; DNA; DNA Adducts - genetics; DNA Adducts - toxicity; DNA, Bacterial - genetics; Escherichia coli; Escherichia coli - genetics; Gels; Genetic mutation; Genetics; Genomes; Humans; Lesions; Malondialdehyde - toxicity; Mops; Mutagenesis, Site-Directed; Mutagenicity; Mutation; Oligonucleotides; Purines - chemistry; Purines - toxicity; Pyrimidines - chemistry; Pyrimidines - toxicity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.16.8652

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-α ]purin-10(3H)-one (M1G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. M1G was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oliogodeoxynucleotide 5′-GGT(M1G)TCCG-3′into a gapped-duplex derivative of the vector. Unmodified and M1G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite M1G resulted in roughly equal numbers of M1G→ A and M1G→ T mutations with few M1G→ C mutations. The total mutation frequency was ≈ 1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite M1G allowed an estimate to be made of the ability of M1G to block replication. The (-)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M1G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M1G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M1G and that M1G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M1G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.