Carcinogen-induced frameshift mutagenesis in repetitive sequences

Abstract

We have constructed plasmids pS3G-1 and pSG4 that contain single acetylaminofluorene adducts within contiguous runs of three (5'-CCCG1G2G3-3') and four (5'-CG1GGG4T-3') guanine residues, respectively. In Escherichia coli, the frequency of induced -1 frameshift mutations was strongly dependent on the position of modification: pS3G-G3 was approximately 100-fold and 10-fold more mutagenic than pS3G-G1 and pS3G-G2, respectively; pSG4-G4 was approximately 600-fold more mutagenic than pSG4-G1. Mutagenesis was SOS-dependent and was markedly reduced in bacteria that were proficient in nucleotide excision repair as compared to a repair-deficient uvrA6 mutant. DNA sequencing showed that -1 frameshift events in pS3G-1 consisted of either targeted mutations (greater than 90% of induced mutations) within the guanine sequence or semitargeted mutations (greater than 10%) in the 5' flanking repetitive cytosine sequence. Semitargeted events, which were observed when acetylaminofluorene modification was at G1 and G2, show that a lesion can reduce the fidelity of replication at positions 5' to its location on the template strand. No semitargeted frameshifts were observed in plasmid pSG4, which lacks a repetitive sequence 5' to the adduct. Our results are consistent with a model for frameshift mutagenesis in which the acetylaminofluorene adduct (i) allows accurate incorporation of cytosine opposite the bulky lesion during DNA synthesis and (ii) impedes elongation of primer/template termini formed opposite the adduct or 5' to the adduct on the template strand, providing increased opportunity for the formation of slipped frameshift intermediates.