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

SP Fink, GR Reddy, LJ Marnett - Proceedings of the …, 1997 - National Acad Sciences
SP Fink, GR Reddy, LJ Marnett
Proceedings of the National Academy of Sciences, 1997National Acad Sciences
The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido [1, 2-α]
purin-10 (3 H)-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 …
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.
National Acad Sciences