Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

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 structure

Structural and biochemical impact of C8-aryl-guanine adducts within the Narl recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

Sherpa Romeo green journal, open accessChemical 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 carbonlinked 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.Ye

A CO2-gas precursor to the March 2015 Villarrica volcano eruption

We present here the first volcanic gas compositional time-series taken prior to a paroxysmal eruption of Villarrica volcano (Chile). Our gas plume observations were obtained using a fully autonomous Multi-component Gas Analyser System (Multi-GAS) in the 3 month-long phase of escalating volcanic activity that culminated into the 3 March 2015 paroxysm, the largest since 1985. Our results demonstrate a temporal evolution of volcanic plume composition, from low CO$_2$/SO$_2$ ratios (0.65-2.7) during November 2014-January 2015 to CO$_2$/SO$_2$ ratios up to ≈ 9 then after. The H$_2$O/CO$_2$ ratio simultaneously declined to <38 in the same temporal interval. We use results of volatile saturation models to demonstrate that this evolution toward CO$_2$-enriched gas was likely caused by unusual supply of deeply sourced gas bubbles. We propose that separate ascent of over-pressured gas bubbles, originating from at least 20-35 MPa pressures, was the driver for activity escalation toward the 3 March climax.This work was funded by the DECADE research initiative of the DCO observatory

Hydroxyl Radical-Induced Oxidation of a Phenolic C-Linked 2′-Deoxyguanosine Adduct Yields a Reactive Catechol

Phenolic toxins stimulate oxidative stress and generate C-linked adducts at the C8-site of 2′-deoxyguanosine (dG). We previously reported that the C-linked adduct 8-(4″-hydroxyphenyl)-dG (<i>p</i>-PhOH-dG) undergoes oxidation in the presence of Na₂IrCl₆ or horseradish peroxidase (HRP)/H₂O₂ to generate polymeric adducts through phenoxyl radical production [Weishar (2008) Org. Lett. 10, 1839−1842]. We now report on reaction of <i>p</i>-PhOH-dG with two radical-generating systems, Cu^II/H₂O₂ or Fe^II-EDTA/H₂O₂, which were utilized to study the fate of the C-linked adduct in the presence of hydroxyl radical (HO^•). The radical-generating systems facilitate (i) hydroxylation of the phenolic ring to afford the catechol adduct 8-(3″,4″-dihydroxyphenyl)-dG (3″,4″-DHPh-dG) and (ii) H-atom abstraction from the sugar moiety to generate the deglycosylated base <i>p</i>-PhOH-G. The ratios of 3″,4″-DHPh-dG to <i>p</i>-PhOH-G were ∼1 for Cu^II/H₂O₂ and ∼0.13 for Fe^II-EDTA/H₂O₂. The formation of 3″,4″-DHPh-dG was found to have important consequences in terms of reactivity. The catechol adduct has a lower oxidation potential than <i>p</i>-PhOH-dG and is sensitive to aqueous basic media, undergoing decomposition to generate a dicarboxylic acid derivative. In the presence of excess <i>N</i>-acetylcysteine (NAC), oxidation of 3″,4″-DHPh-dG produced mono-NAC and di-NAC conjugates. Our results imply that secondary oxidative pathways of phenolic-dG lesions are likely to contribute to toxicity

Utility of 5′‑<i>O</i>‑2,7-Dimethylpixyl for Solid-Phase Synthesis of Oligonucleotides Containing Acid-Sensitive 8‑Aryl-Guanine Adducts

To study the structural and biological impact of 8-aryl-2′-deoxyguanosine adducts, an efficient protocol is required to incorporate them site-specifically into oligonucleotide substrates. Traditional phosphoramidite chemistry using 5′-<i>O</i>-DMT protection can be limiting because 8-aryl-dG adducts suffer from greater rates of acid-catalyzed depurination than dG and are sensitive to the acidic deblock conditions required to remove the DMT group. Herein we show that the 5′-<i>O</i>-2,7-dimethylpixyl (DMPx) protecting group can be used to limit acid exposure and improve DNA synthesis efficiency for DNA substrates containing 8-aryl-dG adducts. Our studies focus on 8-aryl-dG adducts with 8-substituents consisting of furyl (^FurdG), phenyl (^PhdG), 4-cyanophenyl (^CNPhdG), and quinolyl (^QdG). These adducts differ in ring size and sensitivity to acid-promoted deglycosylation. A kinetic study for adduct hydrolysis in 0.1 M aqueous HCl determined that ^FurdG was the most acid-sensitive (55.2-fold > dG), while ^QdG was the most resistant (5.6-fold > dG). The most acid-sensitive ^FurdG was chosen for optimization of solid-phase DNA synthesis. Our studies show that the 5′-<i>O</i>-DMPx group can provide a 4-fold increase in yield compared to 5′-<i>O</i>-DMT for incorporation of ^FurdG into DNA substrates critical for determining adduct impact on DNA synthesis and repair

Enhancing Bulge Stabilization through Linear Extension of C8-Aryl-Guanine Adducts to Promote Polymerase Blockage or Strand Realignment to Produce a C:C Mismatch

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[<i>b</i>]­thienyl-dG ([BTh]­G) and C8-(pyren-1-yl)-dG ([Py]­G), were incorporated into the <i>Nar</i>I (12mer, <i>Nar</i>I­(12) and 22mer, <i>Nar</i>I­(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 <i>Nar</i>I sequence. Primer-elongation assays employing <i>Sulfolobus solfataricus</i> 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 <i>anti</i> and <i>syn</i> 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

Chlorine Functionalization of a Model Phenolic C8-Guanine Adduct Increases Conformational Rigidity and Blocks Extension by a Y‑Family DNA Polymerase

Certain phenoxyl radicals can attach covalently to the C8-site of 2′-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure–activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on <i>in vitro</i> DNA replication by Klenow fragment exo^– (Kf^–) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]­G) and 2,4,6-trichlorophenoxyl radical ([TCP]­G) were inserted into the reiterated G3-position of the <i>Nar</i>I sequence (12-mer, <i>Nar</i>I­(12); and 22-mer, <i>Nar</i>I­(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the <i>Nar</i>I­(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]­G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]­G to afford [PCP]­G does not significantly change the adduct conformation within the <i>Nar</i>I­(12) duplex, as predicted by molecular dynamics simulations. However, when using <i>Nar</i>I­(22) for DNA synthesis <i>in vitro</i>, the chlorinated [PCP]­G and [TCP]­G lesions significantly block DNA replication by Kf^– and Dpo4, whereas [PhO]­G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: Influence of aryl ring size on targeted and semi-targeted mutagenicity

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.ISSN:1362-4962ISSN:0301-561

Structural Influence of C8-Phenoxy-Guanine in the <i>Nar</i>I Recognition DNA Sequence

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 (^PhOG) and C8-4-fluorophenoxy-G (^4FPhOG) were incorporated into the G³ position of the 12-mer <i>Nar</i>I recognition sequence (5′-CT­C­G­G­C­X­C­C­A­TC, where X = G, ^PhOG, or ^4FPhOG) 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 [<i>Nar</i>I′(C)], mismatches with G, A, T [<i>Nar</i>I′(N)], and an abasic site [<i>Nar</i>I′(THF)], and a 10-mer sequence to model a −2 deletion duplex [<i>Nar</i>I′(−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 ¹⁹F nuclear magnetic resonance experiments, and molecular dynamics simulations. On the basis of these findings, ^PhOdG adopts the B conformation opposite C, with the phenoxy moiety residing in the solvent-exposed major groove. However, opposite the G mismatch, ^PhOdG 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 ^PhOG will have a weak mutagenic effect, as determined for the corresponding single-ringed nitrogen-linked C8-dG adduct derived from aniline

Influence of Chlorine Substitution on the Hydrolytic Stability of Biaryl Ether Nucleoside Adducts Produced by Phenolic Toxins

A kinetic study is reported for the acid-catalyzed hydrolysis of oxygen (<i>O</i>)-linked biaryl ether 8-2′-deoxyguanosine (dG) adducts produced by phenolic toxins following metabolism into phenoxyl radical intermediates. Strikingly, the reaction rate of hydrolysis at pH 1 decreases as electron-withdrawing chlorine (Cl) substituents are added to the phenoxyl ring. The Hammett plot for hydrolysis at pH 1 shows a linear negative slope with ρ_X = −0.65, implying that increased Cl-substitution diminishes the rate of hydrolysis by lowering N⁷ basicity. Spectrophotometric titration provided an N⁷H⁺ p<i>K</i>_a value of 1.1 for the unsubstituted adduct 8-phenoxy-dG (Ph-O-dG). Model pyridine compounds suggest N⁷H⁺ p<i>K</i>_a values of 0.92 and 0.37 for 4-Cl-Ph-O-dG and 2,6-dichloro-Ph-O-dG (DCP-O-dG), respectively. Density functional theory (DFT) calculations also highlight the ability of the 8-phenoxy substituent to lower N⁷ basicity and predict a preference for N³-protonation for highly chlorinated <i>O</i>-linked 8-dG adducts in water. The calculations also provide a rationale for the hydrolytic reactivity of <i>O</i>-linked 8-dG adducts in the gas-phase, as determined using electrospray mass spectrometry (ESI-MS). The inclusion of our data now establishes that the order of hydrolytic reactivity at neutral pH for bulky 8-dG adducts is <i>N</i>-linked > <i>C</i>-linked > <i>O</i>-linked, which correlates with their relative ease of N⁷-protonation