SOLUTION STRUCTURE OF THE AMINOFLUORENE-INTERCALATED CONFORMER OF THESYN [AF]-C-8-DG ADDUCT OPPOSITE A -2 DELETION SITE IN THE NARI HOT-SPOT SEQUENCE CONTEXT

This paper addresses structural issues related to the capacity of amin ofluorene [AF] for frameshift mutations of the -2 type on C-8 covalent adduct formation at the G(3) site in the d(C-G(1)-G(2)-C-G(3)-C-C) Na rI hot spot sequence. This problem has been approached from a combined NMR and relaxation matrix analysis computational structural study of the [AF]dG adduct in the d(C-G-G-C-[AF]G-C-C).d(G-G-C-C-G) sequence co ntext at the 12/10-mer adduct level (designated [AF]dG.del(-2) 12/10-m er). The proton spectra of this system are of exceptional quality and are consistent with the formation of an AF-intercalated conformer with the modified guanine in a syn alignment displaced along with the 5'-f lanking cytosine residue into the major groove. The solution structure has been determined by initially incorporating intramolecular and int ermolecular proton-proton distances defined by lower and upper bound d educed from NOESY spectra as restraints in molecular mechanics computa tions in torsion angle space and subsequently refined through restrain ted molecular dynamics calculations based on a NOE distance and intens ity refinement protocol. Strikingly, the [AF]dG.del(-2) 12/10-mer dupl ex adopts only one of two potential AF-intercalation alignments for th e [AF]dG adduct opposite the -2 deletion site in the NarI sequence con text with the extrusion of the dC-[AF]dG step favored completely over extrusion of the [AF]dG-dC step at the lesion site. This polarity esta blishes that the structural perturbation extends 5' rather than 3' to the [AF]dG lesion site in the adduct duplex. This structure of the [AF ]dG adduct opposite a -2 deletion site shows distinct differences with conclusions reported on the alignment of the related acetylaminofluor ene [AAF]dG adduct opposite a -2 deletion site in the identical NarI s equence context [Milhe, C., Fuchs, R. P. P., and Lefevre, J. F. (1996) Eur. J. Biochem. 235, 120-127]. In that study, qualitative NMR data w ithout computational analysis were employed to conclude that the extru sion at the lesion site occurs at the [AAF]dG-dC step for the AAF-inte rcalated conformer of the adduct duplex. The structure of the [AF]dG a dduct opposite a -2 deletion site determined in our group provides mol ecular insights into the architecture of extended slipped mutagenic in termediates involving aromatic amine intercalation and base-displaced syn modified guanines in AF and, by analogy, AAF-induced mutagenesis i n the NarI hot spot sequence context.