Spectroscopic and Theoretical Insights into Sequence Effects of Aminofluorene-Induced Conformational Heterogeneity and Nucleotide Excision Repair^,

Abstract

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.

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

“…The single guanines in these model sequences were adducted with fluoroaminofluorene adduct (dG-FAF). The utility of fluorine-tagged aromatic amine carcinogens as an effective conformational probe has been well documented (10,35,36). As shown in Fig.…”

“…have shown previously (12,32,33,35,39) that purine bases 3Ј to the FAF-adducted site favors the S-conformation, whereas the presence of pyrimidine bases 5Ј to the lesion site favored the major groove B-conformer (36). In full duplexes, a 5Ј-T favors a higher percentage of the major groove B-type conformer, whereas a 5Ј-C favors the stacked conformation.…”

Sequence verification of oligonucleotides containing multiple arylamine modifications by enzymatic digestion and liquid chromatography mass spectrometry (LC/MS)

“…An unmodified dodeca ODN, 5=-CTCGGCGCCATC-3=, was treated with incremental amounts of N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene in a pH 6.0 sodium citrate buffer at 37°C for 18 h [4]. Formation of a covalent adduct at the C8-position of guanine was followed by hydrolysis of the trifluoroacetyl group to yield an AF-modified ODN.…”

Fluorescence Probing of Aminofluorene-Induced Conformational Heterogeneity in DNA Duplexes

“…However, they are not well-suited for studying multiple conformations in dynamic equilibriums, as is the case for AF. As a remedy, we introduced dynamic 19 F nuclear magnetic resonance ( 19 F NMR) spectroscopy coupled with the fluorine probe FAF ( Figure 1A) to study AF-induced conformational heterogeneities in various DNA sequence contexts (7,11,12). Theoretical calculations showed that both the stacking and the electrostatic interactions between the carcinogen and the neighboring base pairs are equally important to the observed sequence effect (12).…”

“…As a remedy, we introduced dynamic 19 F nuclear magnetic resonance ( 19 F NMR) spectroscopy coupled with the fluorine probe FAF ( Figure 1A) to study AF-induced conformational heterogeneities in various DNA sequence contexts (7,11,12). Theoretical calculations showed that both the stacking and the electrostatic interactions between the carcinogen and the neighboring base pairs are equally important to the observed sequence effect (12). 19 F NMR is a powerful tool but requires a relatively large sample, thus making it difficult to apply to all types of carcinogen-induced DNA heterogeneties.…”