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.

We recently reported that the aldehyde residue of an abasic (Ap) site in duplex DNA can generate an interstrand cross-link via reaction with a guanine residue on the opposing strand. This finding is intriguing because the highly deleterious nature of interstrand cross-links suggests that even small amounts of Ap-derived cross-links could make a significant contribution to the biological consequences stemming from the generation of Ap sites in cellular DNA. Incubation of 21-bp duplexes containing a central 5′-CAp sequence under conditions of reductive amination (NaCNBH3, pH 5.2) generated much higher yields of cross-linked DNA than reported previously. At pH 7, in the absence of reducing agents, these Ap-containing duplexes also produced cross-linked duplexes that were readily detected on denaturing polyacrylamide gels. Cross-link formation was not highly sensitive to reaction conditions and, once formed, the cross-link was stable to a variety of work-up conditions. Results of multiple experiments including MALDI-TOF mass spectrometry, gel mobility, methoxyamine capping of the Ap aldehyde, inosine-for-guanine replacement, hydroxyl radical footprinting, and LCMS/MS were consistent with a cross-linking mechanism involving reversible reaction of the Ap aldehyde residue with the N2-amino group of the opposing guanine residue in 5′-CAp sequences to generate hemiaminal, imine, or cyclic hemiaminal cross-links (7-10) that were irreversibly converted under conditions of reductive amination (NaCNBH3/pH 5.2) to a stable amine linkage. Further support for the importance of the exocyclic N2-amino group in this reaction was provided by an experiment showing that installation of a 2-aminopurine-thymine base pair at the cross-linking site produced high yields (15-30%) of a cross-linked duplex at neutral pH, in the absence of NaCNBH3.

The loss of a coding nucleobase from the structure of DNA is a common event that generates an abasic (Ap) site (1). Ap sites exist as an equilibrating mixture of a cyclic hemiacetal and a ring-opened aldehyde. Aldehydes are electrophilic functional groups that can form covalent adducts with nucleophilic sites in DNA. Thus, Ap sites present a potentially reactive aldehyde as part of the internal structure of DNA. Here we report evidence that the aldehyde group of Ap sites in duplex DNA can form a covalent adduct with the N6-amino group of adenine residues on the opposing strand. The resulting interstrand DNA–DNA cross-link occurs at 5′-ApT/5′-AA sequences in remarkably high yields (15–70%) under physiologically relevant conditions. This naturally occurring DNA-templated reaction has the potential to generate cross-links in the genetic material of living cells.

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) is the most abundant food-derived heterocyclic aromatic amine in well-cooked meats and may contribute to the recognized carcinogenicity of processed meats. In this study, a panel of human gut microbes was tested for their ability to convert PhIP to a conjugate PhIP-M1. Eubacterium hallii was newly identified to catalyse the conversion of PhIP to PhIP-M1 with high efficiency. The reaction was shown to involve the metabolism of glycerol to 3-hydroxypropionaldehyde as a key pathway. The proficiency of E. hallii in transforming PhIP in the presence of a complex intestinal microbiota was confirmed using batch fermentations inoculated with effluents from a continuous intestinal fermentation model mimicking human proximal and distal colon microbiota. In batch fermentations inoculated with proximal colon microbiota, PhIP-M1 transformation corresponded to an up to 300-fold increase of E. hallii. In contrast, PhIP transformation of distal colon microbiota was low but increased by 120-fold after supplementation with E. hallii. These findings indicate for the first time the relevance of the abundant commensal strict anaerobe E. hallii in the transformation of a dietary carcinogen that could contribute to its detoxification in the human colon.