F NMR spectroscopy was used to investigate the conformational heterogeneity of two arylamine-modified DNA duplexes, d[CTTCTTG*ACCTC]‚d [GAGGTCAAGAAG], in which G* is either N-(deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl (dG-C8-FABP) (I) or N-(deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (dG-C8-FAF) (II). The 19 F NMR spectrum of I showed a single peak, while that of II revealed two prominent signals with a 55:45 ratio, in good agreement with previous 1 H NMR results (Cho et al. Biochemistry 1992, 31, 9587-9602;1994, 33, 1373-1384. Slow interconversion between the two conformations of II was established by temperature-dependent two-dimensional 19 F NMR chemical exchange spectra. On the basis of magnetic anisotropy effects and isotopic solvent-induced shifts, the 19 F signals at -117.31 and -118.09 ppm in the 19 F NMR spectrum of II were assigned to a relatively undisturbed "B-type" conformer and a highly perturbed "stacked" conformer, respectively. Analysis of the temperature dependent (5-40 °C) line shapes by computer simulation yielded an interconversion barrier (∆G q ) of 14.0 kcal/mol with a chemical exchange time of 2 ms at 30 °C. This new 19 F approach should be very useful in investigating the sequence-dependent conformational heterogeneity of arylamine-modified DNA.

A systematic spectroscopic and computational study was conducted in order to probe the influence of base sequences on stacked (S) versus B-type (B) conformational heterogeneity induced by the major dG adduct derived from the model carcinogen 7-fluoro-2-aminofluorene (FAF). We prepared and characterized eight 12-mer DNA duplexes (-AG*N- series, d[CTTCTAG*NCCTC]; -CG*N- series, d[CTTCTCG*NCCTC]), in which the central guanines (G*) were site-specifically modified with FAF with varying flanking bases (N = G, A, C, T). S/B heterogeneity was examined by CD, UV, and dynamic 19F NMR spectroscopy. All the modified duplexes studied followed a typical dynamic exchange between the S and B conformers in a sequence dependent manner. Specifically, purine bases at the 3'-flanking site promoted the S conformation (G > A > C > T). Simulation analysis showed that the S/B energy barriers were in the 14-16 kcal/mol range. The correlation times (tau = 1/kappa) were found to be in the millisecond range at 20 degrees C. The van der Waals energy force field calculations indicated the importance of the stacking interaction between the carcinogen and neighboring base pairs. Quantum mechanics calculations showed the existence of correlations between the total interaction energies (including electrostatic and solvation effects) and the S/B population ratios. The S/B equilibrium seems to modulate the efficiency of Escherichia coli UvrABC-based nucleotide excision repair in a conformation-specific manner: i.e., greater repair susceptibility for the S over B conformation and for the -AG*N- over the -CG*N- series. The results indicate a novel structure-function relationship, which provides insights into how bulky DNA adducts are accommodated by UvrABC proteins.

Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and -G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1 > G2). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G2∼G1 > G3, a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

Oxidative DNA damage has been shown to involve formation of 8-hydroxy-2'-deoxyguanosine, which may serve as a mispairing lesion during cellular DNA replication. In order to assess the mutagenic potential of this DNA adduct, we examined the possible occurrence of several tautomeric forms and of different base conformations about the deoxyribose. Several spectroscopic and electronic absorption techniques were employed and showed structural changes occurring over a broad pH range. Two pKa's near pH 8 and 12 were observed by pH-solvent partitioning experiments, ultraviolet absorption spectral analyses, and 13C NMR spectroscopic methods. The presence of two pKa's suggested the formation of a dianion, with the second being formed in strong alkali. A comparison of ultraviolet absorption band features of 8-hydroxy-2'-deoxyguanosine with that of different C6,C8-diketo or enol derivatives supported a C8-keto assignment and also provided evidence that this DNA adduct contains a C6-keto group at physiological pH. 13C NMR data showed marked chemical shifts at C6 in solutions of pH 7.4-9.3, indicating the location of the first ionization. Increasing basicity produced further shifts at C5 and C8, indicating the C8 position for the second ionization. Multiple infrared bands were observed in the carbonyl region of the neutral compound, but only a single carbonyl band at 1692 cm-1 remained at pH 9.1, implying a keto group at C8. Determination of the chemical shifts and the nuclear Overhauser enhancements of the N1 and N7 resonances in the proton-decoupled 15N NMR spectrum indicated that both nitrogens were indeed protonated at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Arylamines and polycyclic aromatic hydrocarbons (PAHs), which are known as "bulky" carcinogens, have been studied extensively and upon activation in vivo, react with cellular DNA to form DNA-adducts. The available structure data accumulated thus far has revealed that conformational heterogeneity is a common theme among duplex DNA modified with these carcinogens. Several conformationally diverse structures have been elucidated and found to be in equilibrium in certain cases. The dynamics of the heterogeneity appear to be modulated by the nature of the adduct structure and the base sequences neighboring the lesion site. These can be termed as "adduct- and sequence-induced conformational heterogeneities," respectively. Due to the small energy differences, the population levels of these conformers could readily be altered within the active sites of repair or replicate enzymes. Thus, the complex role of "enzyme-induced conformational heterogeneity" must also be taken into consideration for the establishment of a functional structure-mutation relationship. Ultimately, a major challenge in mutation structural biology is to carry out adduct- and site-specific experiments in a conformationally specific manner within biologically relevant environments. Results from such experiments should provide an accurate account of how a single chemically homogenous adduct gives rise to complex multiple mutations, the earliest step in the induction of cancer.