Sequence Effects on Translesion Synthesis of an Aminofluorene–DNA Adduct: Conformational, Thermodynamic, and Primer Extension Kinetic Studies

The DNA sequence effect is an important structural factor for determining the extent and nature of carcinogen-induced mutational and repair outcomes. In this study, we used two 16-mer template sequences, TG*A [d­(5′-CTTCT<u>TG*A</u>CCTCATTC-3′)] and CG*A [d­(5′-CTTCT<u>CG*A</u>CCTCATTC-3′)], to study the impact of the 5′-flanking nucleotide (T vs C) on aminofluorene (AF)-induced stacked (S)/major groove (B)/wedge (W) conformational heterogeneity during a simulated translesion synthesis. In addition, we probed the sequence effect on nucleotide insertion efficiencies catalyzed by the Klenow fragment (exonuclease-deficient) of DNA polymerase I. Our ¹⁹F NMR/ICD/DSC results showed that AF in the CG*A duplex sequence adopts a greater population of S-conformer than the TG*A sequence. We found that the S conformer of CG*A thermodynamically favors insertion of A over C at the lesion site (<i>n</i>). Significant stalling occurred at both the prelesion (<i>n</i> – 1) and lesion (<i>n</i>) sites; however, the effect was more persistent for the S conformer of CG*A than TG*A at the lesion site (<i>n</i>). Kinetics show that relative nucleotide insertion frequencies (<i>f</i>_ins) were greater for TG*A than the S conformer of CG*A for either dCTP or dATP at the lesion site (<i>n</i>), and the insertion rate was significantly reduced at immediate upstream base pairs (<i>n</i>, <i>n</i> + 1). Taken together, the results provide insight into how the mutagenic AF could exhibit an S/B/W equilibrium in the active site of a polymerase, causing different mutations. This work represents a novel structure–function relationship in which adduct structure is directly linked to nucleotide insertion efficiency in a conformation-specific manner during translesion DNA synthesis

Binary and Ternary Binding Affinities between Exonuclease-Deficient Klenow Fragment (Kf-exo^–) and Various Arylamine DNA Lesions Characterized by Surface Plasmon Resonance

We used surface plasmon resonance (SPR) to characterize the binding interactions between the exonulease-free Klenow fragment (Kf-exo^–) and unmodified and modified dG adducts derived from arylamine carcinogens: fluorinated 2-aminofluorene (FAF), 2-acetylaminofluorene (FAAF), and 4-aminobiphenyl (FABP). Tight polymerase binding was detected with unmodified dG and the correct dCTP. The discrimination of correct versus incorrect nucleotides was pronounced with <i>K</i>_D values in the order of dCTP ≪ dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates with Kf-exo^– binding tighter to the FAAF (<i>k</i>_off: 0.02 s^–1) and FABP (<i>k</i>_off: 0.01 s^–1) lesions than to FAF (<i>k</i>_off: 0.04 s^–1)

Nucleobase-Dependent Reactivity of a Quinone Metabolite of Pentachlorophenol

Pentachlorophenol (PCP) is a possible human carcinogen detected widely in the environment. A quinone metabolite of PCP, tetrachloro-1,4-benzoquinone (Cl4BQ), is a reactive electrophile with the capacity to damage DNA by forming bulky covalent DNA adducts. These quinone adducts may contribute to chlorophenol carcinogenesis, but their structures, occurrence, and biological consequences are not known. Previous studies have indicated that several DNA adducts are formed in vivo in rats exposed to Cl4BQ, but these adducts were not identified structurally. In the present study, we have elucidated the structure of new agent-specific DNA adducts resulting from the reaction of dGuo, dCyd, and Thd with Cl4BQ. These have been characterized chemically by liquid chromatography−electrospray ionization mass spectrometry, HPLC, UV, and NMR analysis. Two dGuo adducts and one dCyd adduct resulting from the reaction of double-stranded DNA with Cl4BQ have been identified. The results indicate that, in the structural context of DNA, Cl4BQ reacts most readily with dGuo compared to the other DNA bases and that the mode of Cl4BQ reactivity is dependent on the base structure; i.e., multiple types of adducts are formed. Finally, DNA adducts consistent with Cl4BQ reactions are observed when DNA or dGuo is treated with PCP and a peroxidase-based bioactivating system

Depurinating Acylfulvene−DNA Adducts: Characterizing Cellular Chemical Reactions of a Selective Antitumor Agent

Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF−DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed