Abstract
Fluorescence spectroscopy was used to study carcinogen-induced conformational heterogeneity in DNA duplexes. The fluorophore 2-aminopurine (AP) was incorporated adjacent (5') to the lesion (G*) in eight different DNA duplexes [d(5'-CTTCT PG* NCCTC-3'):d(5'-GAGGN XTAGAAG-3'), G* = FAF adduct, P = AP, N = G, A, C, T, and X = C, A] modified by FAF [ N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene], a fluorine-tagged model DNA adduct derived from the potent carcinogen 2-aminofluorene. Steady-state measurements showed that fluorescence intensity and Stern-Volmer constants ( Ksv) derived from acrylamide quenching experiments decreased for all carcinogen-modified duplexes relative to the controls, which suggests greater AP stacking in the duplex upon adduct formation. Conformation-specific stacking of AP with the neighboring adduct was evidenced by a sequence-dependent variation in fluorescence intensity, position of emission maximum, degree of emission quenching by acrylamide, and temperature-dependent spectral changes. The magnitude of stacking was in the order of FAF residue in base-displaced stacked (S) > minor groove wedged (W) > major groove B type (B). This work represents a novel utility of AP in probing adduct-induced conformational heterogeneities in DNA duplexes.
Highlights
Conformational heterogeneity is believed to play a role in the structure–function relationships of carcinogen–DNA adducts [1,2]
Steady-state measurements showed that fluorescence intensity and Stern–Volmer constants (Ksv) derived from acrylamide quenching experiments decreased for all carcinogen-modified duplexes relative to the controls, which suggests greater AP stacking in the duplex upon adduct formation
A sequence-dependent decrease in fluorescence intensity was observed for duplexes (I–IV) upon adduct formation
Summary
Conformational heterogeneity is believed to play a role in the structure–function relationships of carcinogen–DNA adducts [1,2]. AF-induced S/B heterogeneity has been implicated in the modulation of sequence-dependent mutational outcomes. N-(2′deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF)-induced S/B/W heterogeneity at the replication fork may influence polymerase function through a long-range distortion effect, contributing to diverse mutational outcomes [10]. AP has been used to examine the structure and dynamics of DNA sequencing [17], abasic sites [18], base flipping [19,20], metal–ion binding [21], and protein–DNA interactions [22], its application for the study of adduct-induced multiple conformers in DNA duplexes is novel. A sequence-dependent variation in the fluorescence properties, such as the position of emission maximum, intensity of fluorescence, and degree of acrylamide quenching, suggested a conformation-specific stacking of AP with the neighboring adduct. The magnitude of stacking was determined to be in the order of FAF residue in S > W > B
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