DNA ADDUCT-INDUCED STABILIZATION OF SLIPPED FRAMESHIFT INTERMEDIATES WITHIN REPETITIVE SEQUENCES - IMPLICATIONS FOR MUTAGENESIS

Chemical carcinogens such as the aromatic amide 2-acetylaminofluorene (AAF) are known to induce -1 frameshift mutation hotspots at repetitiv e sequences. This mutagenesis pathway was suggested to involve slipped intermediates formed during replication. To investigate the stability and structure of such intermediates we have constructed DNA duplexes containing single AAF adducts within a run of three guanine residues. The strand complementary to that bearing the AAF adducts contained eit her the wild-type sequence (homoduplexes) or lacked one cytosine direc tly opposite the run of guanines containing the AAF adduct and thus mo deled the putative slipped mutagenic intermediates (SMIs). The melting temperature of AAF-modified homoduplexes or the unmodified SMI was re duced by almost-equal-to 10-degrees-C relative to the unmodified homod uplex. Surprisingly, AAF adducts stabilized the SMIs as evidenced by a n increase in melting temperature to a level approaching that of the u nmodified homoduplex. The chemical probes hydroxylamine and bromoaceta ldehyde were strongly reactive toward cytosine residues opposite the a dduct in AAF-modified homoduplexes, indicating adduct-induced denatura tion. In contrast, no cytosine reactivities were observed in the AAF-m odified SMIs, suggesting that the two cytosines were paired with unmod ified guanines. Use of diethyl pyrocarbonate to probe the guanine resi dues showed that all three guanines in the unmodified SMI adopted a tr ansient single-stranded state which was delocalized along the repetiti ve sequence. However, when an AAF adduct was present, reduced diethyl pyrocarbonate reactivity at guanines adjacent to the adduct in AAF-mod ified SMIs reflected localization of the bulge to the adducted base. O ur results suggest that AAF exerts a local denaturing and destabilizin g effect within the homoduplex which is alleviated by the formation of a bulge. The stabilization by the AAF adduct of the SMIs may contribu te to the dramatic increase in -1 frameshift mutation frequency induce d by AAF adducts in repetitive sequences.