Structural Alignment of the (+)-trans-anti-Benzo[a]pyrene-dG Adduct Positioned Opposite dC at a DNA Template-Primer Junction ² Binbin Feng, ‡ Andrey Gorin, ‡ Brian E. Hingerty, §,| Nicholas E. Geacintov, ⊥ Suse Broyde, ∇ and Dinshaw J. Patel* ,‡ Cellular Biochemistry and Biophysics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10021, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, Knox Computer Consultants, KnoxVille, Tennessee 37923, and Chemistry and Biology Departments, New York UniVersity, New York, New York 10003 ReceiVed January 13, 1997; ReVised Manuscript ReceiVed August 28, 1997 X ABSTRACT: This study reports on the solution conformation of the covalent (+)-trans-anti-[BP]dG adduct (derived from the binding of the highly mutagenic and tumorigenic (+)-anti-benzo[a]pyrene diol epoxide to the N 2 of deoxyguanosine) positioned opposite dC at a junctional site in the d(A1-A2-C3-[BP]G4-C5- T6-A7-C8-C9-A10-T11-C12-C13)‚d(G14-G15-A16-T17-G18-G19-T20-A21-G22-C23) 13/10-mer DNA sequence. The 13-mer represents the template strand containing the junction [BP]dG4 lesion while the complementary 10-mer models a primer strand which extends upto and is complementary to the modified dG4 residue. The solution conformation has been determined by initially incorporating intramolecular and intermolecular proton-proton distances defined by lower and upper bounds deduced from NOESY spectra as restraints in molecular mechanics computations in torsion angle space and subsequently through restrained molecular dynamics calculations based on a NOE distance and intensity refinement protocol. The duplex segment retains a minimally perturbed B-DNA conformation with all base pairs, including the junctional [BP]dG4‚dC23 pair, in Watson-Crick hydrogen-bonded alignments. The pyrenyl ring is not stacked over the adjacent dC5‚dG22 base pair but is positioned on the minor groove-side of the [BP]dG moiety and directed toward the 5′-end of the template strand. The pyrenyl ring stacks over the base of the non-adjacent dA2 residue in one direction and the sugar ring of dC23 in the other direction. The solution structure of the (+)-trans-anti-[BP]dG adduct opposite dC in the 13/10-mer in which the modified deoxyguanosine adopts an anti glycosidic torsion angle (this study) is in striking contrast to the structure of the same (+)-trans-anti-[BP]dG moiety in a 13/9-mer of the same sequence but without the dC23 residue positioned opposite the adduct site [Cosman, M., et al. (1995) Biochemistry 34, 15334- 15350]. For the latter case, the aromatic portion of the BP residue stacks over the adjacent dC5‚dG22 base pair, the modified deoxyguanosine adopts a syn glycosidic torsion angle and is displaced toward the major groove direction. Insights into the factors that affect the sequence and context dependent conformations of stereoisomeric [BP]dG lesions have emerged following comparison of these two structures with the minor groove conformations of the same (+)-trans-anti-[BP]dG lesion in the fully complementary 11-mer duplex [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918] and in the base displaced-intercalative conformation of the 11/10-mer deletion duplex containing a -1 deletion site opposite the lesion [Cosman, M., et al. (1994) Biochemistry 33, 11507-11517]. The contributing factors where applicable include Watson-Crick base pairing at the site of the lesion, positioning of the carcinogen within the floor of the minor groove, and the tendency of the bulky hydrophobic aromatic BP residue to assume stacked or intercalative conformations. The presence of bulky polycyclic aromatic residues derived from the binding of stereoisomeric mutagenic and tumori- genic metabolites of benzo[a]pyrene to deoxyguanosine residues in DNA, is known to block replication (Reardon et al., 1989; Hruszkewycz et al., 1992; Shibutani et al., 1993), to induce mutations (Wei et al., 1991; Rodriguez & Loechler, 1993), and is believed to constitute the critical intial step in carcinogenesis (Greenblatt et al., 1994; Ross et al., 1995; Denissenko et al., 1996). The most tumorigenic metabolite of the environmental pollutant benzo[a]pyrene (BP) is the (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydroben- zo[a]pyrene [(+)-anti-BPDE] [see reviews by Singer and Grunberger (1983) and Harvey (1991)]. The major adduct resulting from the reaction of (+)-anti-BPDE with native DNA involves the nucleophilic trans-addition of the exo- cyclic amino group of dG residues to C 10 of BPDE. The resulting (+)-trans-anti-[BP]dG moiety has been site-specif- ically incorporated into oligodeoxyribonucleotides of defined sequence for structural studies by NMR methods (Cosman et al., 1992, 1994, 1995; Fountain & Krugh, 1995), by optical spectroscopic techniques (Geacintov et al., 1991; Suh et al., 1995; Ponte´n et al., 1994), as well as for site-specific mutagenesis experiments in ViVo (Mackay et al., 1992; Jelinsky et al., 1995; Moriya et al., 1996). ² This research is supported by NIH Grant CA-46533 to D.J.P., by NIH Grant CA-20851 and DOE Grant DE-FG02-88ER60405 to N.E.G., by NIH Grant CA-28038, NIH Grant RR-06458, and DOE Grant DE- FG02-90ER60931 to S.B., and by DOE Contract DE-AC05-840R21400 with Martin-Marietta Energy Systems and DOE OHER Field Work Proposal ERKP931 to B.E.H. * Corresponding author. ‡ Memorial Sloan Kettering Cancer Center. § Oak Ridge National Laboratory. | Knox Computer Consultants. ⊥ Chemistry Department, NYU. ∇ Biology Department, NYU. X Abstract published in AdVance ACS Abstracts, November 1, 1997. 13769 Biochemistry 1997, 36, 13769-13779 S0006-2960(97)00069-X CCC: $14.00 © 1997 American Chemical Society