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.

First Nations communities in Canada are disproportionately affected by poor water quality. As one example, many communities have been living under boil water advisories for decades, but government interventions to date have had limited impact. This paper examines the importance of using Indigenous research methodologies to address current water issues affecting First Nations. The work is part of larger project applying decolonizing methodologies to Indigenous water governance. Because Indigenous epistemologies are a central component of Indigenous research methods, our analysis begins with presenting a theoretical framework for understanding Indigenous water relations. We then consider three cases of innovative Indigenous research initiatives that demonstrate how water research and policy initiatives can adopt a more Indigenous-centered approach in practice. Cases include (1) an Indigenous Community-Based Health Research Lab that follows a two-eyed seeing philosophy (Saskatchewan); (2) water policy research that uses collective knowledge sharing frameworks to facilitate respectful, non-extractive conversations among Elders and traditional knowledge holders (Ontario); and (3) a long-term community-based research initiative on decolonizing water that is practicing reciprocal learning methodologies (British Columbia, Alberta). By establishing new water governance frameworks informed by Indigenous research methods, the authors hope to promote innovative, adaptable solutions, rooted in Indigenous epistemologies.

Aryl radicals can react at the C8-site of 2'-deoxyguanosine (dG) to produce DNA adducts with a C8-C linkage (denoted C-linked). Such adducts are structurally distinct from those possessing a flexible amine (N-linked) or ether (O-linked) linkage, which separates the C8-aryl moiety from the guanine nucleobase. In the current study, two model C-linked C8-dG adducts, namely, C8-benzo[b]thienyl-dG ([BTh]G) and C8-(pyren-1-yl)-dG ([Py]G), were incorporated into the NarI (12mer, NarI(12) and 22mer, NarI(22)) hotspot sequence for frameshift mutations in bacteria. For the first time, C-linked C8-dG adducts are shown to stabilize the -2 deletion duplex within the NarI sequence. Primer-elongation assays employing Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) demonstrates the influence of C8-aryl ring size and shape in promoting Dpo4 blockage or strand realignment to produce a C:C mismatch downstream of the adduct site. Molecular dynamics simulations of the -2 deletion duplex suggest that both anti and syn adduct structures are energetically accessible. These findings provide a rationale for describing the biochemical outcome induced by C-linked C8-dG adducts when processed by Dpo4.

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Phenoxyl radicals can covalently attach to the C8 site of 2'-deoxyguanosine (dG) to generate oxygen-linked biaryl ether C8-dG adducts. To assess the structural impact of an O-linked C8-dG adduct in duplex DNA, C8-phenoxy-G ((PhO)G) and C8-4-fluorophenoxy-G ((4FPhO)G) were incorporated into the G(3) position of the 12-mer NarI recognition sequence (5'-CTCGGCXCCATC, where X = G, (PhO)G, or (4FPhO)G) using solid-phase DNA synthesis with O-linked C8-dG phosphoramidites. The modified strands were hybridized to six different complementary strands that include regular base pairing to C [NarI'(C)], mismatches with G, A, T [NarI'(N)], and an abasic site [NarI'(THF)], and a 10-mer sequence to model a -2 deletion duplex [NarI'(-2)]. All duplex structures were characterized using UV-vis thermal melting temperature analysis, and in each instance, the O-linked C8-phenoxy-G adducts were found to destabilize the duplex relative to the unmodified controls. The most stable duplex structures match the O-linked C8-dG adduct against C and a G mismatch, which are comparable in terms of stability. These duplexes were further characterized using circular dichroism, dynamic (19)F nuclear magnetic resonance experiments, and molecular dynamics simulations. On the basis of these findings, (PhO)dG adopts the B conformation opposite C, with the phenoxy moiety residing in the solvent-exposed major groove. However, opposite the G mismatch, (PhO)dG adopts a "W-type" wedge conformation with the phenoxy group residing in the minor groove. These studies predict that the O-linked C8-dG lesion (PhO)G will have a weak mutagenic effect, as determined for the corresponding single-ringed nitrogen-linked C8-dG adduct derived from aniline.