Mutation spectra of Glu-P-1 in Salmonella: induction of hotspot frameshifts and site-specific base substitutions.

Abstract

We used colony probe hybridization and PCR/DNA sequence analysis to determine the mutations in approximately 1,640 revertants of the -1 frameshift allele hisD3052 and approximately 260 revertants of the base substitution allele hisG46 of Salmonella typhimurium induced by the heterocyclic amine cooked food mutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1). All of the mutations were at sites containing guanine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100% of the Glu-P-1-induced mutations at the frameshift allele in strains TA1978 (uvr+) and TA1538 (delta uvrB) and 99% in TA98 (delta uvrB, pKM101). To explain the induction of these hotspot mutations by Glu-P-1, we describe here a more detailed version of our recently proposed correct incorporation/slippage model [Genetics:136:731, 1994]. We propose that after cytosine is incorporated correctly opposite a Glu-P-1-adducted guanine, various slipped intermediates may form (a total of 18), depending on which guanine is adducted and whether it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hotspot sequence by Glu-P-1. Although the pKM101 plasmid does not influence the mutagenic potency or mutational spectrum of Glu-P-1 at the frameshift allele, it is required by Glu-P-1 to revert the base substitution allele, where Glu-P-1 induces G-C --> T-A transversions (75%) and G-C --> tA-T transitions (25%) exclusively at a single site (the second position of the CCC codon of the hisG46 allele). The limited (20-30 times less) base substitution mutagenic potency of Glu-P-1 relative to its frameshift mutagenic potency as well as the extreme site specificity exhibited by Glu-P-1 for base substitutions may have bearing on the lack of base substitutions identified in ras genes in Glu-P-1-induced rat colon tumors.