MUTATION SPECTRA OF GLU-P-1 IN SALMONELLA - INDUCTION OF HOTSPOT FRAMESHIFTS AND SITE-SPECIFIC BASE SUBSTITUTIONS

We used colony probe hybridization and PCR/ DNA sequence analysis to d etermine the mutations in similar to 1,640 revertants of the -1 frames hift allele hisD3052 and similar to 260 revertants of the base substit ution allele hisG46 of Salmonella typhimurium induced by the heterocyc lic amine cooked food mutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]i midazole (Glu-P-1). All of the mutations were at sites containing guan ine, which is the base at which Glu-P-1 forms DNA adducts. A hotspot m utation involving the deletion of a CG or GC within the sequence CGCGC GCG accounted for 100% of the Glu-P-1-induced mutations at the framesh ift allele in strains TA1978 (uvr(+)) and TA1538 (Delta uvrB) and 99% in TA98 (Delta uvrB, pKM101). To explain the induction of these hotspo t mutations by Glu-P-1, we describe here a more detailed version of ou r 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 whet her it remains within the helix or becomes extrahelical. This variety of mutational pathways may account for the high mutability of the hots pot sequence by Glu-P-1. Although the pKM101 plasmid does not influenc e the mutagenic potency or mutational spectrum of Glu-P-1 at the frame shift allele, it is required by Glu-P-1 to revert the base substitutio n 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 ti mes less) base substitution mutagenic potency of Glu-P-1 relative to i ts frameshift mutagenic potency as well as the extreme site specificit y 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. (C) 1994 Wiley-Liss, Inc.