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.

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DNA sustains a wide variety of damage, such as the formation of abasic sites, pyrimidine dimers, alkylation adducts, or oxidative lesions, upon exposure to UV radiation, alkylating agents, or oxidative conditions. Since such damage may be acutely toxic or mutagenic and potentially carcinogenic, it is of interest to gain insight into how their structures impact biochemical processing of DNA, such as synthesis, transcription, and repair. Lesion-specific molecular probes have been used to study polymerase-mediated translesion DNA synthesis of abasic sites and TT dimers, while other probes have been developed to specifically investigate the alkylation adduct O 6 -Bn-G and the oxidative lesion 8-oxo-G. In this review recent examples of lesion-specific molecular probes are surveyed; their specificities of incorporation opposite target lesions compared to unmodified nucleotides are discussed, and limitations of their applications under physiologically relevant conditions are assessed.Within years of the discovery that DNA 1 was responsible for transmitting hereditary information (1), and before publication of the double helical structure of DNA (2), researchers began to probe its susceptibility to various types of damage (3). Exposure of DNA to UV radiation, alkylating agents, and reactive oxidative species leads to the formation of abasic sites, pyrimidine dimers, alkylation adducts, and oxidative damage products. Each of these types of lesions can be mutagenic, sometimes carcinogenic, and this chemical process, therefore, is highly relevant to cellular biology and disease. For example, UV radiation damageinduced skin cancer was estimated to result in over one million new cases of the disease in the United States in 2008 (4). Further, DNA alkylation by tobacco-derived carcinogens contributes to lung cancer, which is estimated will result in about 160,000 deaths in the United States in 2009 (5,6). These examples of the biological consequences of DNA damage underscore the continued need for understanding the underlying molecular mechanisms.

Two mixed ligand complexes of ruthenium(ii) [Ru(bzimpy)(bpy)(OH(2))](2+) (1) and [Ru(bzimpy)(phen)(OH(2))](2+) (2) have been synthesized and characterized by FAB mass, (1)H NMR, cyclic voltammetry and spectroelectrochemical measurements. Controlled potential electrolysis of these complexes results in the conversion of ruthenium(ii) to ruthenium(iii) at 0.6 V and ruthenium(iii) to ruthenium(iv) at 0.8 V vs. SCE. The binding constant of these complexes with DNA has been determined electrochemically and found to be (3.58 +/- 0.25) x 10(4) and (2.87+/- 0.2) x 10(4) M(-1). Viscosity measurements suggest that these complexes bind with DNA through intercalation. Such intercalative binding to DNA has been found to induce chirality to the two complexes. Electrochemically generated ruthenium(iv) species of these complexes have been found to bring about oxidative cleavage in DNA.

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'-CTTCTTG*ACCTCATTC-3')] and CG*A [d(5'-CTTCTCG*ACCTCATTC-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 (19)F 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 (n). Significant stalling occurred at both the prelesion (n - 1) and lesion (n) sites; however, the effect was more persistent for the S conformer of CG*A than TG*A at the lesion site (n). Kinetics show that relative nucleotide insertion frequencies (f(ins)) were greater for TG*A than the S conformer of CG*A for either dCTP or dATP at the lesion site (n), and the insertion rate was significantly reduced at immediate upstream base pairs (n, n + 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.

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We used surface plasmon resonance (SPR) to characterize the binding interactions between 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 KD values in order of dCTP << dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates with Kf-exo- binding tighter to the FAAF (koff: 0.02s-1) and FABP (koff: 0.01s-1) lesions than to FAF (koff: 0.04s-1).

Background: DNA lesions in different sequence contexts exhibit alternate conformations. Results: DNA adduct conformation is differentially modulated by DNA structure, DNA polymerase, and the presence of an incoming nucleoside triphosphate. Conclusion: Adduct conformation is influenced by binding of a non-hydrolysable nucleotide to pol ␤ but not Klenow fragment. Significance: Adduct conformation and coding potential is modulated by constraints imposed by the polymerase active site.