Search

Minor adducts, derived from the covalent binding of anti-benzo(a)pyrene-7,8-dihydroxy-9,- 10-epoxide to cellular DNA, may play an important role in generating mutations and initiating cancer. We have applied a combined NMR-computational approach including intensity based refinement to determine the solution structure of the minor (+)-cis-anti-(BP)dA adduct positioned opposite dT in the d(C1-T2-C3-T4-C5-(BP)A6-C7-T8-T9-C10-C11)‚(d(G12-G13-A14-A15-G16-T17-G18- A19-G20-A21-G22) 11-mer duplex. The BP ring system is intercalated toward the 5'-side of the (BP)- dA6 lesion site without disrupting the flanking Watson-Crick dC5‚dG18 and (BP)dA6‚dT17 base pairs. This structure of the (+)-cis-anti-(BP)dA‚dT 11-mer duplex, containing a bay region benzo(a)pyrenyl (BP)dA adduct, is compared with the corresponding structure of the (+)-trans-anti-(BPh)dA‚dT 11-mer duplex (Cosman et al., Biochemistry 32, 12488-12497, 1993), which contains a fjord region benzo(c)- phenanthrenyl (BPh)dA adduct with the same R stereochemistry at the linkage site. The carcinogen intercalates toward the 5'-direction of the modified strand in both duplexes (the adduct is embedded within the same sequence context) with the buckling of the Watson-Crick (BP)dA6‚dT17 base pair more pronounced in the (+)-cis-anti-(BP)dA‚dT 11-mer duplex compared to its Watson-Crick (BPh)dA‚dT17 base pair in the (+)-trans-anti-(BPh)dA‚dT 11-mer duplex. The available structural studies of covalent polycyclic aromatic hydrocarbon (PAH) carcinogen-DNA adducts point toward the emergence of a general theme where distinct alignments are adopted by PAH adducts covalently linked to the N 6 of adenine when compared to the N 2 of guanine in DNA duplexes. The (BPh)dA and (BP)dA N 6 -adenine adducts intercalate their polycyclic aromatic rings into the helix without disruption of their modified base pairs. This may reflect the potential flexibility associated with the positioning of the covalent tether and the benzylic ring of the carcinogen in the sterically spacious major groove. By contrast, such an intercalation without modified base pair disruption option appears not to be available to (BP)dG N 2 -guanine adducts where the covalent tether and the benzylic ring are positioned in the more sterically crowded minor groove. In the case of (BP)dG adducts, the benzopyrenyl ring is either positioned in the minor groove without base pair disruption, or if intercalated into the helix, requires disruption of the modified base pair and displacement of the bases out of the helix. Benzo(a)pyrene is an ubiquitous environmental precar- cinogen produced by incomplete combustion of fossil fuels

The Escherichia coli genome contains a C-G1-G2-C-G3-C-C NarI hot spot sequence for -2 deletion mutations at G3 by aromatic amine carcinogens 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) that form covalent adducts at the C8-position of the guanine ring. Each of the three guanines are positioned in different sequence contexts (C-G1-G, G-G2-C, and C-G3-C) which provides an opportunity to investigate the potential sequence dependent interconversion between AF-intercalated and AF-external conformers of the [AF]dG adduct positioned opposite dC within the NarI sequence at the duplex level. We have prepared and purified DNA duplexes containing the [AF]dG adduct positioned in C-[AF]G-G, G-[AF]G-C, and C-[AF]G-C NarI sequence contexts and observe the ratio of AF-intercalated to AF-external conformers to be 30:70, 10:90, and 50:50, respectively. We have applied a combined NMR-molecular mechanics approach to define the structure of the AF-external conformer in the G-[AF]G-C NarI sequence context where it is the predominant conformation (90%) in solution. The modified guanine of the [AF]dG adduct aligns through Watson-Crick pairing with its partner cytosine and is stacked into the helix between flanking Watson-Crick dG.dC base pairs. The AF-external conformer with its anti-[AF]dG residue causes minimal perturbations in the DNA duplex at and adjacent to the lesion site with the covalently linked fluorenyl ring readily accommodated in the major groove and tilted toward the 5'-end of the modified strand of the helix. This paper on the structure of the AF-external conformer with an anti-[AF]dG adduct together with the preceding paper in this issue on the structure of the AF-intercalated conformer with a syn-[AF]dG adduct defines for the first time the capacity of the mutagenic [AF]dG lesion to adopt interconverting syn and antialignments with the equilibrium shifting between the conformers depending on nearest neighbor and next-nearest neighbor sequences. Perhaps, recognition of the [AF]dG lesion by the repair machinery would be able to discriminate between the AF-intercalated conformer with its base displacement-fluorenyl ring insertion perturbation of the helix and the AF-external conformer where the DNA helix is essentially unperturbed at the lesion site and the fluorenyl ring is positioned with directionality in the major groove.

The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S), 10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino group of guanine residues in DNA in vivo and in vitro. While the (-)-7S,8R,9R,10Senantiomer, (-)-anti-BPDE, also reacts with DNA to form similar covalent N2-deoxyguanosyl adducts, this diol epoxide is nontumorigenic and its mutagenic activities are different from those of (+)-anti-BPDE. In this work, T4 ligase-induced cyclization methods have been employed to demonstrate that the (+)-anti-[BP]-N2-dG lesions (G*) cause significantly greater amounts of bending and circularization of the one-base overhang undecamer duplex 5'-d(CACAT[G*]TACAC).d(TGTACATGTGG) than the stereoisomeric oligonucleotide duplex with G* = (-)-anti-[BP]-N2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotides, the ratio of circular to linear DNA multimers reaches values of 8-9 for circle contour sizes of 99-121 base pairs, while for the (-)-anti-[BP]-N2-dG-modified DNA this ratio reaches a maximum value of only approximately 1 at 154-176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90-92% of the observed circular ligation products range from 30 to 51 degrees per (+)-trans-anti-[BP]-N2-dG lesion and from 20 to 40 degrees per (-)-trans-anti-[BP]-N2-dG lesion. In the case of unmodified DNA, the probability of circular product formation is at least 1 order of magnitude less efficient than in the BPDE-modified sequences and about 90% of the circular products exhibit bending angles in the range of 14 -19 degrees . In the most abundant circular products observed experimentally, the bending angles are 40 degrees and 26 +/- 2 degrees per (+)-anti-[BP]- or (-)-anti-[BP]-modified 11-mer; these values correspond to a net contribution of 21-26 degrees and 5-19 degrees , respectively, to the observed overall bending per lesion. The coexistence of circular DNA molecules of different sizes and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends, which permit efficient cyclization, especially in the case of (+)-trans-[BP]-N2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N2-dG lesion in the 11-mer duplex 5'-d(CACAT[G*]TACAC).d(GTGTACATGTG) indicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the duplex on the 5'-side of the modified guanosine residue, which may be due to bending enhanced base pair opening and bending induced by the bulky carcinogen residue. The implications of base sequence-dependent flexibilities and conformational mobilities of anti-[BP]-N2-dG lesions on DNA replication and mutation are discussed.

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.

This paper reports on the solution structure of the [AF]dG adduct positioned opposite a deletion site in a DNA oligomer duplex that defines the alignment of the covalent aminofluorene--C8-guanine adduct relative to the deletion site. The combined NMR molecular mechanics computational studies were undertaken on the [AF]dG adduct embedded in the d(C5-[AF]G6-C7).d(G16-G17) sequence context in a duplex containing 11 residues on the modified strand and 10 on the partner, with no base opposite the modification. The exchangeable and nonexchangeable protons of the aminofluorene moiety and the nucleic acid were assigned following analysis of two-dimensional NMR data sets in H2O and D2O solution. The solution conformation of the [AF]G.del 11-mer duplex has been determined by 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. The aminofluorene ring of [AF]dG6 is intercalated between intact Watson-Crick dC5.dG17 and dC7.dG16 base pairs with the guanine base of [AF]dG6 in a syn alignment displaced into the major groove. The syn glycosidic torsion angle at [AF]dG6 is supported by both carbon and proton chemical shift data for the sugar resonances of the modified guanine residue. The long axis of the aminofluorene ring is parallel to the long axis of the flanking dG.dC base pairs with the AF ring undergoing rapid 180 degrees flips on the NMR time scale. The intercalation site is wedge shaped with a pronounced propeller-twisting and buckling of the dC5.dG17 base pair. The guanine base of [AF]dG6, which is positioned in the major groove, is inclined relative to the helix axis and stacks over the 5'-flanking dC5 residue in the solution structure. The intercalative-base displacement structure of the [AF]dG.del 11-mer duplex exhibits several unusually shifted proton resonances that can be readily accounted for by the ring current contributions of the guanine purine and carcinogen fluorene aromatic rings of the [AF]dG6 adduct. There are similarities between this structure of the AF-C8-dG covalent adduct positioned opposite a deletion site and the (+)-trans-anti-BP-N2-dG covalent adduct positioned opposite a deletion site in the same sequence context reported previously from this laboratory [Cosman et al. (1994) Biochemistry 33, 11507-11517]. The chromophores are intercalated into the helix opposite the deletion site with displacement of the modified guanine into the major groove in both cases.(ABSTRACT TRUNCATED AT 400 WORDS)

The tumorigenic metabolite of benzo[a]pyrene, the (+)-7R,8S,9S,10R enantiomer, and the nontumorigenic mirror-image isomer, (-)-7S,8R,9R, 10S, of r7,t8-dihydroxy-t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE) bind covalently to the exocyclic amino group of deoxyguanosine (N2-dG) in native DNA. These adducts can cause structural perturbations such as DNA bends, which in turn may influence the cellular processing of these lesions. The characteristics of bends in site-specifically modified oligodeoxyribonucleotide duplexes induced by single (+)- and (-)-anti-[BP]-N2-dG lesions were examined by self-ligation and gel electrophoresis techniques. The modified residues (dG*) were centrally positioned in the 11-mer oligonucleotide d(CACAXG*XACAC) complexed with the natural complementary strands, with X = T or C, or in oligonucleotides 16 or 22 base pairs long with the same centrally positioned 11-mer. Among the four stereochemically distinct lesions, the 10S(+)-trans-anti-[BP]-N2-dG adducts were significantly more bent than any of the other three stereoisomeric adducts and were selected for detailed studies. In the TG*T sequence context (X = T), the retardation factor RL (apparent length of multimer/sequence length) is approximately independent of the phasing (distance, in base pairs, between the lesions) of the adducts with respect to the helical repeat (10.5 base pairs/helix turn). In contrast, in the CG*C sequence context (X = C), RL is markedly lower in the case of ligated 16-mers than in the case of ligated 11-mer duplexes. The dependence of RL on the phasing of the bends as a function of the helical repeat, indicate that the bends associated with (+)-trans-anti-[BP]-N2-dG lesions are relatively rigid in the d(...CG*C...).d(...GCG...) sequences, and flexible in the d(...TG*T...).d(...ACA...) sequence context. These differences are attributed to the orientations of the pyrenyl residues on the 5'-side of the modified deoxyguanosine residues in the minor groove and to the intrinsic roll and tilt characteristics of DNA dinucleotide steps CG, GC, TG, and GT. The influence of flanking bases on the extent and character of DNA bending suggest that base sequence effects may be important in the cellular processing of (+)-trans-anti-[BP]-N2-dG lesions.

We report below on a conformational equilibrium between AF-intercalated and AF-external states in slow exchange for the [AF]dG lesion positioned opposite dC in the d(C-[AF]G-C).d(G-C-G) sequence context. The slow exchange between states is attributed to interconversion between syn glycosidic torsion angle in the AF-intercalated and anti torsion angle in AF-external conformers of the [AF]dG opposite dC containing duplex. The present paper describes an NMR-molecular mechanics study that defines the solution structure of the AF-intercalated conformer for the case of [AF]dG adduct positioned opposite dC in the d(C-[AF]G-C).d(G-C-G) sequence context. The structure is of the base displacement-intercalation type where the aminofluorene ring is intercalated into the helix between intact Watson-Crick dG.dC base pairs, which results in a displacement of the modified guanine ring into the major groove where it stacks with the major groove edge of its 5'-flanking cytosine in the adduct duplex. The conformational equilibrium between AF-intercalated conformer (approximately 70%) with a syn alignment and AF-external conformer (approximately 30%) with an anti alignment for the [AF]dG adduct positioned opposite dC in the d(C-[AF]G-C).d(G-C-G) sequence context can be contrasted with our earlier demonstration that the population is 100% for the AP-intercalated conformer with a synalignment at the N-(deoxyguanosin-8-yl)-2-aminopyrene ([AP]dG) adduct site positioned opposite dC in the same sequence context [Mao, B., Vyas, R. R., Hingerty, B. E., Broyde, S., Basu, A. K., and Patel, D. J. (1996) Biochemistry, 35, 12659-12670]. This shift in population may reflect the much larger size of the pyrenyl ring of the [AP]dG adduct compared to the fluorenyl ring of the [AF]dG adduct which in turn might provide for a greater overlap of the aromatic amine with the flanking base pairs in the intercalated conformer of the former adduct in DNA.

A solution structural study has been undertaken on the aminofluorene-C8-dG ([AF]dG) adduct located at a single strand-double strand d(A1-A2-C3-[AF]G4-C5-T6-A7-C8-C9-A10-T11-C12-C13).d (G14-G15-A16-T17-G18-G19-T20-A 21-G22) 13/9-mer junction (designated [AF]dG 13/9-mer) using proton-proton distance and intensity restraints derived from NMR data in combination with a computational protocol, which includes intensity refinement. This single strand-double strand junction models one arm of a replication fork composed of a 13-mer template strand, which contains the [AF]dG modification site, and a 9-mer primer strand, which has been elongated up to, but not including, the modified guanine. The NMR data establish that the duplex segment retains a minimally perturbed B-DNA conformation including Watson-Crick hydrogen-bonding at the junctional dC5.dG22 base pair. The NMR spectra are consistent with the guanine ring of the [AF]dG4 adduct adopting a syn glycosidic torsion angle and being displaced into the major groove with the adjacent dC3 residue displaced into the minor groove. Such a base displacement of the modified guanine is accompanied by stacking of one face of the fluorene ring of [AF]dG4 with the dC5.dG22 base pair, while the other face of the flourene ring is stacked with the purine ring of the nonadjacent dA2 residue in the intensity-refined solution structures of the [AF]dG 13/9-mer. A comparison of structural features of the C8-[AF]dG adduct (this study) with those of the (+)-trans-anti-N2-[BP]dG adduct [Cosman et al. (1995) Biochemistry 34, 15334-15350] in the same 13/9-mer junctional sequence context has identified common features associated with the alignment of the modified guanine adducts at the template-primer junction. Thus, despite differences in the covalent linkage site for the C8-[AF]dG and (+)-trans-anti-N2-[BP]dG adducts, one face of the aromatic ring of the carcinogen stacks over the junctional base pair and in so doing displaces the modified guanine in a syn alignment into the major groove. These results lend credence to earlier proposals that such an adduct alignment may represent a common mutagenic conformer at a template-primer junction associated with a replication fork.

The deoxyribooligonucleotide 5'-d(CTCACATGTACACTCT) was reacted separately with the chiral diol epoxide isomers 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE)] and 7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-anti-BPDE)], to produce the modified oligonucleotides 5'-d(CTCACATGBPDETACACTCT). Adducts in which either (+)-anti-BPDE or (-)-anti-BPDE are covalently bound via their C10 positions by trans addition to the exocyclic amino group of the single G residues were isolated and purified by HPLC methods. Snake venom phosphodiesterase (SVPD, phosphodiesterase I), which hydrolyzes DNA from the 3'-OH terminus to the 5'-end, digests the (+)-trans-anti-BPDE-oligonucleotide adducts at a significantly faster rate than that of the sterically different (-)-trans-anti-BPDE-oligonucleotide adducts. However, using spleen phosphodiesterase (SPD, phosphodiesterase II), which hydrolyzes DNA in the 5'-->3' direction, the opposite stereoselective resistance to digestion is observed. Using shorter BPDE-modified oligonucleotides as standards, the enzyme stall sites have been defined by gel electrophoresis methods; the most digestion-resistant phosphodiester linkage is the 5'-d(...T-G*...)-3' bond in the case of (+)-trans-BPDE-modified oligonucleotide adducts for both enzymes, SVPD and SPD (the starred G denotes the site of BPDE modification). In the case of the (-)-trans-BPDE-modified oligonucleotide adducts, the phosphodiester bond on the 3'-side of the modified G [5'-d(...G*-T...)-3'] is most resistant to digestion by both enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)