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(3 H )-one (M 1 G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli . M 1 G was placed at position 6256 in the (−)-strand of M13MB102 by ligating the oligodeoxynucleotide 5′-GGT(M 1 G)TCCG-3′ into a gapped-duplex derivative of the vector. Unmodified and M 1 G-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 M 1 G resulted in roughly equal numbers of M 1 G→A and M 1 G→T mutations with few M 1 G→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 M 1 G allowed an estimate to be made of the ability of M 1 G to block replication. The (−)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M 1 G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M 1 G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M 1 G and that M 1 G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M 1 G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.