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

Fink SP, Reddy GR, Marnett LJ
Proc Natl Acad Sci U S A. 1997 94 (16): 8652-7

PMID: 9238032 · PMCID: PMC23062 · DOI:10.1073/pnas.94.16.8652

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-alpha]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 approximately 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.

MeSH Terms (9)

DNA, Bacterial DNA Adducts Escherichia coli Humans Malondialdehyde Mutagenesis, Site-Directed Mutation Purines Pyrimidines

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