Abstract
Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G3-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo− (Kf−) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.
Highlights
The genetic integrity of a living cell is constantly assaulted by various chemical species reacting with DNA
When the modified NarI[12] oligonucleotides were paired opposite strands containing an opposing G in order to analyse the degree of G:G mismatch stabilization, the C8-aryl-dG adducts had variable influences on duplex stability
The smallest FurG lesion is the most flexible and exhibits greater accessibility to the syn conformation when paired opposite C. (ii) When the modified NarI[12] sequences were hybridized with the truncated 10-mer sequence [–2], i.e. mimicking a slipped mutagenic intermediate (SMI), none of the C-linked C8-substituted -deoxyguanosine (C8-dG) adducts stabilized the truncated duplex compared to the unmodified control, suggesting the inability of these lesions to induce –2 frameshift mutations. (iii) Primer extension reactions with an adducted NarI[22] template:15mer primer catalyzed by the high-fidelity replicative polymerase E. coli pol I Klenow fragment exo− (Kf−), indicated that increased C8-aryl ring size inhibits error-prone synthesis by Kf−
Summary
The structurally related aromatic organic carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) [7,8,9], arylhydrazines [10,11,12] and phenols [13,14,15,16], may undergo bioactivation, producing aryl radical species that covalently attach to the C8-site of dG to produce carbon-linked C8aryl-dG adducts In these cases, the adducts are similar in structure to their N-linked counterparts in terms of aryl ring size and shape, but do not have an amine linkage, thereby altering the orientation of the modification in the DNA duplex as well as reducing conformational flexibility. In contrast to N-linked C8-dG adducts, there is limited understanding of the relationship between the struc-
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