Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl-Guanine Lesion in the <i>Nar</i>I Mutational Hotspot: Evidence for Enhanced <i>Syn</i> Adduct Formation

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Nitroaromatic compounds represent a major class of industrial chemicals that are also found in nature. Polycyclic derivatives are regarded as potent mutagens and carcinogens following bioactivation to produce nitrenium electrophiles that covalently modify DNA to afford N-linked C8-2′-deoxyguanosine (C8-dG) lesions that can induce frameshift mutations, especially in CpG repeat sequences. In contrast, their monocyclic counterparts typically exhibit weak mutagenicity or a lack thereof, despite also undergoing bioactivation to afford N-linked C8-dG adducts. Recently, it has been reported that cyano substitution can greatly increase the mutagenicity of nitroaniline derivatives that are components of azo dyes. The basis of this "cyano effect" may be rooted in the formation of a novel polycyclic adduct arising from initial formation of the N-linked C8-dG adduct followed by a cyclization process involving N7 of dG and the ortho-CN group of the attached C8aryl moiety to generate a quinazolinimine ring as part of a fused tetracyclic C8,N7-dG adduct structure. The present work structurally characterizes this novel cyclic adduct using a combination of optical spectroscopies, NMR analysis, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Our data indicate that this highly fluorescent cyclic adduct adopts the promutagenic syn conformation and can stabilize the slipped mutagenic intermediate (SMI) within the CpG repeat of the NarI sequence, which is a hotspot for frameshift mutagenesis mediated by polycyclic N-linked C8-dG adducts. In contrast, the open para-CN (4-aminobenzontrile-derived) N-linked C8-dG adduct is less likely to disrupt the canonical B-form. Together, our results provide a rationale for the potent mutagenicity of cyano-substituted nitroaniline derivatives recently reported in frameshift-sensitive tester strains.

Section: ■ Experimental Proceduresmentioning

confidence: 99%

Structure of an Unusual Tetracyclic Deoxyguanosine Adduct: Implications for Frameshift Mutagenicity of ortho-Cyano Nitroanilines

Manning

Al‐Abdul‐Wahid

Manderville

et al. 2019

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“…The main objective of the present study was to determine the basis of how sequence context influences the efficiency and fidelity of Kf – -mediated primer extension of oligonucleotide templates containing the C-linked FBP-dG lesion within CG-dinucleotide repeat sequences, which are hotspots for −2 frameshift mutations. , We previously demonstrated the capacity of FBP-dG to stabilize the SMI within the Nar I sequence and exhibit emission sensitivity to SMI formation . Specifically, FBP-dG displayed a 4-fold increase in emission intensity at 420 nm following excitation at 315 nm within the SMI versus the FP duplex.…”

Section: Discussionmentioning

confidence: 94%

“…The 22-mer adducted oligonucleotide sequences ( Nar I: 5′-AT CGGCXC CATCCCTTACGAGC-3′ and CG 3 : 5′-AT GCGCXC CATCCCTTACGAGC-3′, X = FBP-dG) were prepared on a BioAutomation MerMade 12 automatic DNA synthesizer using standard and the FBP-dG modified phosphoramidite. Full synthetic details of the FBP-dG phosphoramidite have been described previously . Following synthesis, oligonucleotides were cleaved from the solid support and deprotected using 2 mL of 30% ammonium hydroxide solution at 55 °C for 12 h and purified by RP-HPLC.…”

Section: Methodsmentioning

confidence: 99%

“…Melting temperatures ( T m ) of the duplexes (3 μM) were determined by variable-temperature UV analysis using a Cary 300-Bio UV–vis spectrophotometer equipped with a 6 × 6 Multicell Peltier block-heater using Hellma 114-QS 10 mm light path cells. T m values were determined as previously outlined . All measurements were performed three times, and the mean T m values are reported.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we developed a C-linked C8-biphenyl-dG adduct analog bearing a fluorine atom (FBP-dG) with a turn-on emission response suited for monitoring the formation of SMI structures mediated by polymerase processing of adducted templates . The adduct strongly favors the syn conformation in the fully paired (FP) duplex and stabilizes the SMI compared to an unmodified two-nucleotide bulge by 6–7 °C; the largest Δ T m for the SMI containing a C-linked C8-dG adduct that we have observed to date.…”

Section: Introductionmentioning

confidence: 99%

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Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion

Berger

Manderville

Sturla

2019

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Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2′-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5′-GGCXCC-3′, where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (−2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats (NarI: 3′-CXCGGC-5′ and CG 3 : 3′-CXCGCG-5′, X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo − (Kf − ) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf − extended the primer past FBP-dG within a CG 3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on −2 frameshift mutations mediated by the C-linked FBP-dG lesion.

Effects of a Second Local DNA Damage Event on the Toxicity of the Human Carcinogen 4-Aminobiphenyl: A Molecular Dynamics Study of a Damaged DNA Structure

Kung

Takyi

Wetmore

2022

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Exposure of humans to carcinogenic aromatic amines (AAs) occurs daily. AAs are bioactivated in cells into products that attack DNA, primarily leading to N-linked C8-dG adducts. Previous work on DNA containing a single AA-derived adduct (monoadducted DNA) has shown a structure–function relationship between the damaged DNA conformation and cellular outcomes. However, relatively little is known about the conformation and biological outcomes of DNA containing two bulky adducts (diadducted DNA) in close proximity. To fill this current void in the literature, the present work uses quintuplet 0.5 μs MD simulations to understand the structural impact of DNA exposure to the potent bladder carcinogen 4-aminobiphenyl (ABP), which is found in cigarette smoke and select dyes, and results in the widely studied N-linked ABPdG adduct. Specifically, 18 unique DNA duplexes were investigated that contain one or two ABPdG adducts in the anti and/or syn glycosidic orientation(s) in all combinations of three G positions in the NarI mutation hotspot for AAs (5′-G1G2CG3CC). Monoadducted DNA displays sequence-dependent conformational heterogeneity, with the G1 site having the greatest anti preference, and highlights the range of helical structures associated with the syn lesion orientation [i.e., stacked (S), intercalated (I), and wedge (W) conformations]. Diadducted DNA results in interesting lesion separation effects on the conformational heterogeneity, including a greater anti preference for neighboring adducts (G1G2) and a greater syn preference for next-nearest neighbor damaged sites (G2G3) compared to monoadducted DNA. As a result, an increase in the number of ABPdG adducts changes the conformational heterogeneity of ABP-exposed DNA depending on the relative positions of the lesions and thereby could result in increased or decreased toxicity upon human exposure to elevated levels of ABP.