1ax6

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SOLUTION STRUCTURE OF THE [AF]-C8-DG ADDUCT OPPOSITE A-2 DELETION SITE IN THE NARI HOT SPOT SEQUENCE CONTEXT; NMR, 6 STRUCTURES

Overview

This paper addresses structural issues related to the capacity of, aminofluorene [AF] for frameshift mutations of the -2 type on C8 covalent, adduct formation at the G3 site in the d(C-G1-G2-C-G3-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.

About this Structure

1AX6 is a Protein complex structure of sequences from [1] with AF as ligand. Full crystallographic information is available from OCA.

Reference

Solution structure of the aminofluorene-intercalated conformer of the syn [AF]-C8-dG adduct opposite a--2 deletion site in the NarI hot spot sequence context., Mao B, Gorin A, Gu Z, Hingerty BE, Broyde S, Patel DJ, Biochemistry. 1997 Nov 25;36(47):14479-90. PMID:9398167

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