Conformational analysis of the 2′‐deoxyribofuranose ring from proton–proton coupling constants: Analysis of a nucleoside‐carcinogen adduct formed from 2‐acetylaminofluorene utilizing a three‐state model

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The conformation and dynamics of the dinucleotide d-CpG modified at the C(8) position of the guanine ring by the carcinogen 2-(acetylamino)fluorene has been investigated by high-field 1H NMR spectroscopy. A two-state analysis of chemical shift data has enabled estimation of the extent of intramolecular stacking in aqueous solution as a function of temperature. The stacking, which is mostly fluorene-cytosine, is virtually complete in the low-temperature range. The 500-MHz 1H NMR spectrum consists of two subspectra near ambient temperatures due to a 14.3 +/- 0.3 kcal/mol barrier to internal rotation about the amide bond in the stacked form. A large barrier to internal rotation about the guanyl-nitrogen bond at C(8) has also been ascertained, but separate NMR subspectra were not detected due to the predominance of one of the torsional diastereomers (alpha' = 90 degrees) in the fully stacked state. Problems of self-association and chemical exchange were identified and overcome to enable analysis of the sugar-phosphate backbone conformation utilizing coupling constants. For the exocyclic C(4')-C(5') bond of the deoxyguanosine moiety, there is a high gauche+ (gamma = 60 degrees) conformer population, which is uncommon for a purine nucleotide with a syn orientation about the glycosyl bond. The gauche- conformation (gamma = 300 degrees), which is normally present in syn purine nucleotides in solution, was not detected. The exocyclic C(5')-O(5') torsion of the deoxy-guanosine moiety remains near the classical energy minimum (beta = 180 degrees) in the major stacked conformations. The sugar ring of the deoxycytidine moiety is predominantly in the C2'-endo conformation, while the deoxyguanosine ring is a mixture of conformations, one of which appears to be unusually puckered. The results support intercalation models of modified DNA and suggest a looped-out structure, with the modified guanine being the first base in the loop. Such structures could explain the relatively rapid rate of repair and the frame-shift mutations of this type of adduct.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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NMR study of stacking interactions and conformational adjustments in the dinucleotide-carcinogen adduct 2'-deoxycytidylyl-(3 .fwdarw. 5)-2'-deoxy-8-(N-fluoren-2-ylacetamido)guanosine

Evans¹,

Levine²

1988

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“…The task of understanding how structurally similar arylamine-DNA adducts can lead to different mutagenicities and repair rates will require three-dimensional structural details of the modification sites. Although NMR methods have been used extensively for this purpose (Evans et al, 1980(Evans et al, , 1984Evans & Miller, 1982;Evans & Levine, 1987Sharma & Box, 1985;Shapiro et al, 1986), the number of reported solution NMR studies of intact arylamine-DNA adducts at the duplex level is small (Norman et al, 1989;Cho et al, 1992;O'Handley et al, 1993). This is due in part to the spectral complexities encountered in the proton NMR spectra of modified duplex oligomers (Harris et al, 1988) and to the technical difficulties in preparing carcinogen-modified oligonucleotides (Basu & Essigmann, 1988).…”

mentioning

confidence: 99%

NMR structural studies of a 15-mer DNA duplex from a ras protooncogene modified with the carcinogen 2-aminofluorene: conformational heterogeneity

Cho¹,

Beland²,

Marques³

1994

120

Proton NMR studies were conducted on the complementary 15-mer DNA duplex, d(5'-TACTCTTCTT[AF]GACCT).d (5'-AGGTCAAGAAGAGTA) (designated as the AF-modified duplex). The sequence represents a portion of the mouse c-Ha-ras protooncogene and was selectively modified to contain a single N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) adduct at the deoxyguanosine corresponding to the first base of codon 61. The AF-modified duplex was found to exist in multiple conformations, with one being predominant (approximately 60%). The exchangeable and nonexchangeable protons belonging to the major conformer were sufficiently well-resolved to allow the assignment of the majority of the base and sugar protons. The one-dimensional proton spectra, as well as the NOE cross-peak patterns associated with this conformer of the AF-modified duplex both in H2O and D2O spectra, were strikingly similar to those observed for the major conformer of an analogous duplex containing N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) in the same position [Cho, B.P., Beland, F. A., & Marques, M. M. (1992) Biochemistry 31, 9587-9602]. The experimental results suggest that the AF- and ABP-modified duplexes adopt essentially identical major conformations, with each arylamine moiety being positioned in the major groove of a slightly disturbed B-type DNA duplex. Nonetheless, the absence of specific NOE cross peaks in the vicinity of the modification site indicates that the local structural perturbation is more severe in the AF-modified duplex. Although insufficient data precluded a detailed characterization of the minor conformers of the AF-modified duplex, the observation of significant shielding of the AF aromatic protons suggests a more dramatic structural alteration at the adduct site, possibly involving extensive stacking with the neighboring bases. The higher content (30-40%) of the minor conformers observed for the AF-modified duplex contrasted with the low contribution (5-10%) of similar structures in the ABP-modified duplex and may be attributed to a better overlapping efficiency of the planar AF ring with the nearby bases. Since the significant local perturbation observed in the minor conformers could provide a possible mechanism for mutations, our results support the view that the structural differences in the arylamine fragments of otherwise identical adducts have a direct influence on the conformational heterogeneities, which in turn may play a significant role in arylamine carcinogenesis.

“…High-resolution proton NMR methods have been applied to elucidate structural features of AAF-G adducts at the mononucleotide (Evans & Levine, 1987), dinucleotide (Evans & Levine, 1988), and hexanucleotide (Sharma & Box, 1985) single-strand level and at the nonanucleotide duplex level (Krugh et al, 1986). The latter NMR study addresses questions related to the conformation of (AAF)G opposite cytidine in the center of a 9-mer duplex (Krugh et al, 1986).…”

mentioning

confidence: 99%

NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].cntdot.A[anti] pair formation and its pH dependence

Norman¹,

Abuaf²,

Hingerty³

et al. 1989

140

162

This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-A17-G18- A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 degrees C in H2O and D2O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn].A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5.G18 and C7.G16 base pairs on either side of the (AF)G6[syn].A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7].[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand.(ABSTRACT TRUNCATED AT 400 WORDS)