Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde

Abstract

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 oligodeoxynucleotide 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.