MUTAGENICITY IN ESCHERICHIA-COLI OF THE MAJOR DNA ADDUCT DERIVED FROMTHE ENDOGENOUS MUTAGEN MALONDIALDEHYDE

The spectrum of mutations induced by the naturally occurring DNA adduc e pyrimido[1,2-alpha]purin-10(3H)-one (M(1)G) was determined by site-s pecific approaches using M13 vectors replicated in Escherichia coli, M le was placed at position 6256 in the (-)-strand of M13MB102 by ligati ng the oligodeoxynucleotide 5'-GGT(M(1)G)TCCG-3' into a gapped-duplex derivative of the vector, Unmodified and M(1)G-modified genomes contai ning either a cytosine or thymine at position 6256 of the (+)-strand w ere transformed into repair-proficient and repair-deficient E. coli st rains, and base pair substitutions were quantitated by hybridization a nalysis, Modified genomes containing a cytosine opposite M(1)G resulte d in roughly equal numbers of M(1)G-->A and M(1)G-->T mutations with f ew M(1)G-->C mutations. The total mutation frequency was approximate t o 1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a t hymine 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 b y M(1)G was 18%, Experiments using E. coli with different genetic back grounds 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 excisi on repair complex. These studies indicate that M(1)G, which is present endogenously in DNA of healthy human beings, is a strong block to rep lication and an efficient premutagenic lesion.