Solution structure of the aminofluorene-intercalated conformer of the syn [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 aminofluorene [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) NarI 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 context at the 12/10-mer adduct level (designated [AF]dG.del(-2) 12/10-mer). 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'-flanking cytosine residue into the major groove. The solution structure has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bound deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently refined through restrainted molecular dynamics calculations based on a NOE distance and intensity refinement protocol. Strikingly, the [AF]dG.del(-2) 12/10-mer duplex adopts only one of two potential AF-intercalation alignments for the [AF]dG adduct opposite the -2 deletion site in the NarI sequence context 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 establishes 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 acetylaminofluorene [AAF]dG adduct opposite a -2 deletion site in the identical NarI sequence context [Milhe, C., Fuchs, R. P. P., and Lefevre, J. F. (1996) Eur. J. Biochem. 235, 120-127]. In that study, qualitative NMR data without computational analysis were employed to conclude that the extrusion at the lesion site occurs at the [AAF]dG-dC step for the AAF-intercalated conformer of the adduct duplex. The structure of the [AF]dG adduct opposite a -2 deletion site determined in our group provides molecular insights into the architecture of extended slipped mutagenic intermediates involving aromatic amine intercalation and base-displaced syn modified guanines in AF and, by analogy, AAF-induced mutagenesis in the NarI hot spot sequence context.