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 ²,‡ Bing Mao, § Andrey Gorin, § Zhengtian Gu, § Brian E. Hingerty, | Suse Broyde, ⊥ and Dinshaw J. Patel* ,§ Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, Life Sciences DiVision, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, and Biology Department, New York UniVersity, New York, New York 10003 ReceiVed September 5, 1997 X ABSTRACT: 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. The aromatic amine carcinogens 2-acetylaminofluorene [AAF] and 2-aminofluorene [AF] form covalent adducts following metabolic or chemical activation at the C 8 position of guanine on DNA (reviewed in ref 1). The more bulky [AAF]dG adduct differs only in the presence of an acetyl group linked to the N 2 fluorenyl moiety in the place of the hydrogen in the [AF]dG adduct 1 (Figure 1A). This substitution is responsible for the observed mutagenic differences produced by these two adducts, which in turn has provoked the decades long interest in the structural basis for their distinct mutagenic spectrum of activities. The application of NMR methods has provided consider- able insights into the structure of [AF]dG adducts as a function of base or absence of it on the partner strand opposite the lesion site and as a function of sequence context flanking the lesion site. Initial studies on [AF]dG adducts positioned opposite dA (2) and dG (3) established that the modified guanine in a syn alignment stacked into the helix with the aminofluorene positioned within the walls of the minor groove and directed toward the partner strand. Structural studies by Eckel and Krugh (4, 5) established a slow interconversion between AF-intercalated and AF- external conformers for the [AF]dG adduct positioned opposite dC at the duplex level. These authors established that the [AF]dG adduct adopts an anti alignment for the AF- external conformer and also favored an anti alignment for the AF-intercalated conformer in the equilibrium. This conformational equilibrium between AF-intercalated and AF- external conformers in slow exchange has also been observed in other sequence contexts (6-8). However, a fundamental ² This research is supported by NIH Grant CA-49982 to D.J.P., by NIH Grants CA-28038 and CA-75449, NIH Grant RR-06458, and DOE Grant DE-FG02-90ER60931 to S.B., and by DOE Contract DE-AC05- 960R22464 with Lockheed Martin Energy Research and DOE OHER Field Work Proposal ERKP931 to B.E.H. ‡ The coordinates of the [AF]dG‚del(-2) 12/10-mer have being deposited in the Brookhaven Protein Data Base (file name 1AX6). § MemorialSloan-Kettering Cancer Center. | Oak Ridge National Laboratory. ⊥ New York University. X Abstract published in AdVance ACS Abstracts, November 15, 1997. 14479 Biochemistry 1997, 36, 14479-14490 S0006-2960(97)02205-8 CCC: $14.00 © 1997 American Chemical Society