NMR DATA SHOW THAT THE CARCINOGEN N-2-ACETYLAMINOFLUORENE STABILIZES AN INTERMEDIATE OF -2 FRAMESHIFT MUTAGENESIS IN A REGION OF HIGH MUTATION FREQUENCY

The heteroduplex, d(ACCGGCGCCACA). d(TGTGG-CCGGT), containing two bulg ed bases, a cytosine and the guanine G7, either unmodified or modified with the carcinogen N-2-acetylaminofluorene, have been studied by NMR as models of slipped-mutagenic intermediates (SMI). The melting tempe rature of the modified heteroduplex is strongly increased compared wit h that of the unmodified heteroduplex, NMR studies have shown that all the bases of the unmodified heteroduplex are stacked within the helix , without any disruption of the sequential connectivities. The two str ands are in a B-like conformation. Nevertheless, exchangeable-proton s tudies have revealed that base pairing is very weak, or even lacking, over two base pairs apart from the bulge. Concerning the modified hete roduplex, no B-like connectivity is observed in the G5-C9 segment. Mor eover, the cytosine C8 is rejected outside the helix, whereas the N-2- acetylaminofluorene moiety is inserted within the helix. The G5 . C18, C6 . G17 and C9 . G16 bases are remarkably stable when the temperatur e is increased, in agreement with the high melting temperature. Some s mall unassigned peaks reveal the presence of the minor conformation in equilibrium. The strong stabilisation of the N-2-acetylaminofluorene- modified heteroduplex compared with the unmodified duplex is in agreem ent with the high N-2-acetylaminofluorene-induced mutation frequency c ompared with the spontaneous frequency and with the hypothesis of muta genesis occurring during replication.