Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde.
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.
Fink
S P
SP
Department of Biochemistry, Center in Molecular Toxicology and The Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
Reddy
G R
GR
Marnett
L J
LJ
D018736:DNA Adducts / Q000235:genetics / Q000633:toxicity*; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008315:Malondialdehyde / Q000633:toxicity*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011687:Purines / Q000737:chemistry / Q000633:toxicity*; D011743:Pyrimidines / Q000737:chemistry / Q000633:toxicity*
D018736:DNA Adducts; D004269:DNA, Bacterial; D011687:Purines; D011743:Pyrimidines; C107643:pyrimido(1,2-a)purin-10(3H)-one; D008315:Malondialdehyde