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Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA. Adduct formation proceeds via the reaction of the amino group(s) on ethidium with the ring-opened aldehyde residue of the AP site in DNA to yield an imine. Ethidium-AP adducts may form under a variety of circumstances due to the ubiquitous occurrence of AP sites in cellular and synthetic DNA.

2-Methylfuran (2-MF) is a well-known industrial chemical and also formed via thermal treatment of food. One main source of 2-MF in the human diet is coffee. 2-MF is known to form 3-acetylacrolein (AcA, 4-oxopent-2-enal) via cytochrome P 450 metabolism and further reacts with amino acids in vivo. Still the reactivity toward other biomolecules is rather scarce. Therefore, AcA was synthesized, and its reaction with 2'-deoxyadenosine (dA), 2'deoxyguanosine (dG), 2'deoxycytosine (dC), and 2'-deoxythymidine (dT) was tested. For this purpose, adduct formation was performed by acid hydrolysis of 2,5-dihydro-2,5-dimethoxy-2-methylfuran (DHDMMF) as well as pure AcA. The structures of these adducts were confirmed by UPLC-ESI + -MS/MS fragmentation patterns and 1 H-/ 13 CNMR spectra. Except for dT, which showed no reactivity, all adducts of AcA were characterized, which enabled the development of sensitive quantification methods via (U)HPLC-ESI ± -MS/MS. Pure AcA was synthesized by oxidation of 2-MF using dimethyldioxirane (DMDO), and its behavior in aqueous medium was studied. Incubations of AcA and isolated DNA of primary rat hepatocytes (pRH) showed time- and dose-dependent formation of the identified DNA adducts dA-AcA, dG-AcA, or dC-AcA. In contrast, the DNA adducts dA-AcA, dG-AcA, or dC-AcA were not detected on a cellular level when pRH were incubated with 2-MF or AcA. This indicates an efficient detoxification or reaction with biomolecules in the cell, although the induction of other DNA damage, possibly also by other metabolites, cannot be ruled out in principle.

Aristolochic acids are one of the major compounds in aristolochia plants, which are nephrotoxic and carcinogenic. A method was established for the detection and identification of aristolochic acids and their DNA adducts in four different herbs using ultra-high performance liquid chromatography-ion mobility quadrupole time-of flight mass spectrometry. Solid phase extraction conditions were optimized to improve the sensitivity of the experiment by using 40 mg of C 18 as adsorbent and 100 μL ethanol as elution solvent. At a collision energy of 10-40 eV, these compounds and cleavage patterns were precisely identified and analyzed by secondary fragmentation and collision cross section values. The obtained mass spectrometry data were then analyzed by targeted metabolomics, including principal component analysis, partial least squares-discriminant analysis and hierarchical clustering analysis, and importing the samples in the established model, the confidence values can reach 0.61 and 0.76. All in all, this method can provide a useful tool for the detection of aristolochic acids and deoxyribonucleic acid adducts. In conclusion, this method was successfully used for the detection and identification of aristolochic acids and their DNA adducts., Competing Interests: Declaration of competing interest The authors (Ying Shi, Yi-Wen Wu, Qian-Xue Shen, Jun Cao) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

DNA lesions are linked to cancer, aging, and various diseases. The recognition and sequencing of special DNA lesions are of great interest but highly challenging. In this paper, an unnatural-base-pair-promoting method for sequencing highly mutagenic ethenodeoxycytidine (εC) DNA lesions that occurred frequently is developed. First, a promising unnatural base pair of dεC-dNaM to recognize εC lesions is identified, and then a conversion PCR is developed to site-precise transfer dεC-dNaM to dTPT3-dNaM for convenient Sanger sequencing. The low sequence dependence of this method and its capacity for the enrichment of dεC in the abundance of as low as 1.6 × 10 -6 nucleotides is also validated. Importantly, the current method can be smoothly applied for recognition, amplification, enrichment, and sequencing of the real biological samples in which εC lesions are generated in vitro or in vivo, thus offering the first sequencing methodology of εC lesions at single-base resolution. Owing to its simple operations and no destruction of inherent structures of DNA, the unnatural-base-pair strategy may provide a new platform to produce general tools for the sequencing of DNA lesions that are hardly sequenced by traditional strategies., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Utilizing molecular dynamics and free energy perturbation, we examine the relative binding affinity of several covalent polycyclic aromatic hydrocarbon - DNA (PAH-DNA) adducts at the central adenine of NRAS codon-61, a mutational hotspot implicated in cancer risk. Several PAHs classified by the International Agency for Research on Cancer as probable, possible, or unclassifiable as to carcinogenicity are found to have greater binding affinity than the known carcinogen, benzo[a]pyrene (B[a]P). van der Waals interactions between the intercalated PAH and neighboring nucleobases, and minimal disruption of the DNA duplex drive increases in binding affinity. PAH-DNA adducts may be repaired by global genomic nucleotide excision repair (GG-NER), hence we also compute relative free energies of complexation of PAH-DNA adducts with RAD4-RAD23 (the yeast ortholog of human XPC-RAD23) which constitutes the recognition step in GG-NER. PAH-DNA adducts exhibiting the greatest DNA binding affinity also exhibit the least RAD4-RAD23 complexation affinity and are thus predicted to resist the GG-NER machinery, contributing to their genotoxic potential. In particular, the fjord region PAHs dibenzo[a,l]pyrene, benzo[g]chrysene, and benzo[c]phenanthrene are found to have greater binding affinity while having weaker RAD4-RAD23 complexation affinity than their respective bay region analogs B[a]P, chrysene, and phenanthrene. We also find that the bay region PAHs dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, and dibenzo[a,h]anthracene exhibit greater binding affinity and weaker RAD4-RAD23 complexation affinity than B[a]P. Thus, the study of PAH genotoxicity likely needs to be substantially broadened, with implications for public policy and the health sciences. This approach can be broadly applied to assess factors contributing to the genotoxicity of other unclassified compounds., Competing Interests: Competing interests statement:NSF grant CHE-2203366.

Genotoxicants originating from inflammation, diet, and environment can covalently modify DNA, possibly initiating the process of carcinogenesis. DNA adducts have been known for long, but the old methods allowed to target only a few known DNA adducts at a time, not providing a global picture of the "DNA adductome". DNA adductomics is a new research field, aiming to screen for unknown DNA adducts by high resolution mass spectrometry (HRMS). However, DNA adductomics presents several analytical challenges such as the need for high sensitivity and for the development of effective screening approaches to identify novel DNA adducts. In this work, a sensitive untargeted DNA adductomics method was developed by using ultra-high performance liquid chromatography (UHPLC) coupled via an ESI source to a quadrupole-time of flight mass spectrometric instrumentation. Mobile phases with ammonium bicarbonate gave the best signal enhancement. The MS capillary voltage, cone voltage, and detector voltage had most effect on the response of the DNA adducts. A low adsorption vial was selected for reducing analyte loss. Hybrid surface-coated analytical columns were tested for reducing adsorption of the DNA adducts. The optimized method was applied to analyse DNA adducts in calf thymus, cat colon, and human colon DNA by performing a MS E acquisition (all-ion fragmentation acquisition) and screening for the loss of deoxyribose and the nucleobase fragment ions. Fifty-four DNA adducts were tentatively identified, hereof 38 never reported before. This is the first untargeted DNA adductomics study on human colon tissue, and one of the few untargeted DNA adductomics studies in the literature reporting the identification of such a high number of unknowns. This demonstrates promising results for the application of this sensitive method in future human studies for investigating novel potential cancer-causing factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

It is generally accepted that adjacent guanine residues in DNA are the primary target for platinum antitumor drugs and that differences in the conformations of the Pt-DNA adducts can play a role in their antitumor activity. In this study, we investigated the effect of the carrier ligand cis -1,3-diaminocyclohexane ( cis -1,3-DACH) upon formation, stability, and stereochemistry of the ( cis -1,3-DACH)PtG 2 and ( cis -1,3-DACH)Pt(d(GpG)) adducts (G = 9-EthlyGuanine, guanosine, 5'- and 3'-guanosine monophosphate; d(GpG) = deoxyguanosil(3'-5')deoxyguanosine). A peculiar feature of the cis -1,3-DACH carrier ligand is the steric bulk of the diamine, which is asymmetric with respect to the Pt-coordination plane. The ( cis -1,3-DACH)Pt(5'GMP) 2 and ( cis -1,3-DACH)Pt(3'GMP) 2 adducts show preference for the ΛHT and ∆HT conformations, respectively (HT stands for Head-to-Tail). Moreover, the increased intensity of the circular dichroism signals in the cis -1,3-DACH derivatives with respect to the analogous cis -(NH 3 ) 2 species could be a consequence of the greater bite angle of the cis -1,3-DACH carrier ligand with respect to cis -(NH 3 ) 2 . Finally, the ( cis -1,3-DACH)Pt(d(GpG)) adduct is present in two isomeric forms, each one giving a pair of H8 resonances linked by a NOE cross peak. The two isomers were formed in comparable amounts and had a dominance of the HH conformer but with some contribution of the ΔHT conformer which is related to the HH conformer by having the 3'-G base flipped with respect to the 5'-G residue.

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSA MYR ) and its AA complexes (HSA MYR /AA-I and HSA MYR /AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 10 6  M -1 for HSA/AA-I versus 8.4 and 9.0 · 10 5  M -1 for HSA/AA-II. Fluorescence quenching of the HSA Trp 214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The clinically used antihypertensive agent hydralazine rapidly generates hydrazone-derived adducts by reaction with apurinic/apyrimidinic (also known as abasic or AP) sites in many different sequences of duplex DNA. The reaction rates are comparable to those of some AP-trapping reagents previously described as "ultrafast." Initially, reversible formation of a hydrazone adduct is followed by an oxidative cyclization reaction that generates a chemically stable triazolo[3,4- a ]phthalazine adduct. The net result is that the reaction of hydralazine with AP sites in duplex DNA yields a rapid and irreversible adduct formation. Although the hydrazone and triazolo[3,4- a ]phthalazine adducts differ by only two mass units, it was possible to use MALDI-TOF-MS and ESI-QTOF-nanospray-MS to quantitatively characterize mixtures of these adducts by deconvolution of overlapping isotope envelopes. Reactions of hydralazine with the endogenous ketone pyruvate do not prevent the formation of the hydralazine-AP adducts, providing further evidence that these adducts have the potential to form in cellular DNA. AP sites are ubiquitous in cellular DNA, and rapid, irreversible adduct formation by hydralazine could be relevant to the pathogenesis of systemic drug-induced lupus erythematosus experienced by some patients. Finally, hydralazine might be developed as a probe for the detection of AP sites, the study of cellular BER, and marking the location of AP sites in DNA-sequencing analyses.

Safrole is a natural product present in many plants and plant products, including spices and essential oils. During cellular metabolism, it converts to a highly reactive trans-isosafrole (SF) intermediate that reacts with genomic DNA and forms N 2 -SF-dG and N 6 -SF-dA DNA adducts, which are detected in the oral tissue of cancer patients with betel quid chewing history. To study the SF-induced carcinogenesis and to probe the role of low fidelity translesion synthesis (TLS) polymerases in bypassing SF adducts, herein, we report the synthesis of N 2 -SF-dG modified DNAs using phosphoramidite chemistry. The N 2 -SF-dG modification in the duplex DNA does not affect the thermal stability and retains the B-form of helical conformation, indicating that this adduct may escape the radar of common DNA repair mechanisms. Primer extension studies showed that the N 2 -SF-dG adduct is bypassed by human TLS polymerases hpolκ and hpolη, which perform error-free replication across this adduct. Furthermore, molecular modeling and dynamics studies revealed that the adduct reorients to pair with the incoming nucleotide, thus allowing the effective bypass. Overall, the results indicate that hpolκ and hpolη do not distinguish the N 2 -SF-dG adduct, suggesting that they may not be involved in the safrole-induced carcinogenicity.

DNA base damage is a major source of oncogenic mutations 1 . Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation 2 . Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication 3,4 , we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts 5 . The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution., (© 2024. The Author(s).)

Apurinic/apyrimidinic (AP) sites, 5-formyluracil (fU) and 5-formylcytosine (fC) are abundant DNA modifications that share aldehyde-type reactivity. Here, we demonstrate that polyamines featuring at least one secondary 1,2-diamine fragment in combination with aromatic units form covalent DNA adducts upon reaction with AP sites (with concomitant cleavage of the AP strand), fU and, to a lesser extent, fC residues. Using small-molecule mimics of AP site and fU, we show that reaction of secondary 1,2-diamines with AP sites leads to the formation of unprecedented 3'-tetrahydrofuro[2,3,4-ef]-1,4-diazepane ('ribodiazepane') scaffold, whereas the reaction with fU produces cationic 2,3-dihydro-1,4-diazepinium adducts via uracil ring opening. The reactivity of polyamines towards AP sites versus fU and fC can be tuned by modulating their chemical structure and pH of the reaction medium, enabling up to 20-fold chemoselectivity for AP sites with respect to fU and fC. This reaction is efficient in near-physiological conditions at low-micromolar concentration of polyamines and tolerant to the presence of a large excess of unmodified DNA. Remarkably, 3'-ribodiazepane adducts are chemically stable and resistant to the action of apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphoesterase 1 (TDP1), two DNA repair enzymes known to cleanse a variety of 3' end-blocking DNA lesions., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Acrolein is a major component in cigarette smoke and a product of endogenous lipid peroxidation. It is difficult to distinguish human exposure to acrolein from exogenous sources versus endogenous causes, as components in cigarette smoke can stimulate lipid peroxidation in vivo. Therefore, analysis of acrolein-induced DNA and protein adducts by the highly accurate, sensitive, and specific mass spectrometry-based methods is vital to estimate the degree of damage by this IARC Group 2A carcinogen. This Perspective reviews the analyses of acrolein-induced DNA and protein adducts in humans by mass spectrometry focusing on samples accessible for biomonitoring, including DNA from leukocytes and oral cells and abundant proteins from blood, i.e., hemoglobin and serum albumin.

Rationale: Acrylamide is classified as a probable human carcinogen that is metabolised to glycidamide, which can covalently bind to DNA. The aim of this study was to investigate the formation of N7-glycidamide guanine (N7-GA-Gua) adducts in human blood DNA following exposure to acrylamide present in carbohydrate-rich foods as part of the normal human diet., Methods: Lymphocyte DNA was extracted from blood samples obtained from healthy human volunteers. Following thermal depurination of the DNA samples, N7-GA-Gua adducts were quantified using a validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method incorporating a stable isotope labelled internal standard. Estimated dietary acrylamide intake was recorded by completion of food frequency questionnaires for the 24 hours prior to volunteer blood donation., Results: An LC/MS/MS method was validated with a limit of detection of 0.25 fmol and a lower limit of quantitation of 0.50 fmol on column. N7-GA-Gua adducts were detected in human blood DNA with the levels ranging between 0.3 to 6.3 adducts per 10 8 nucleotides. The acrylamide intake was calculated from the food frequency questionnaires ranging between 20.0 and 78.6 μg., Conclusions: Identification and quantification of N7-GA-Gua adducts in the blood DNA of healthy volunteers suggests that dietary acrylamide exposure may lead to the formation of DNA adducts. This important finding warrants further investigation to ascertain a correlation between environmental/dietary acrylamide exposure and levels of DNA adducts., (© 2021 John Wiley & Sons Ltd.)

DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum-acridinylthiourea conjugate [PtCl(en)(L)](NO 3 ) 2 (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5'-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5'-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KF exo- ) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO 3 ) 2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine).

Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1, N 6 -ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5'/3' flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

ADP-ribosyltransferases use NAD + to catalyse substrate ADP-ribosylation 1 , and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria 2-4 . Reversible ADP-ribosylation has traditionally been considered a protein-specific modification 5 , but recent in vitro studies have suggested nucleic acids as targets 6-9 . Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa) 10 . We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Rationale: As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by liquid chromatography/mass spectrometry. This method is based on the tendency of adducted nucleobases to locally disrupt the DNA structure (forming a "DNA bubble") potentially increasing exposure of their nucleophilic (including active hydrogen) sites for preferential alkylation. Also encouraging this strategy is that the scope of nucleotide excision repair is very broad, and this system primarily recognizes DNA bubbles., Methods: A cationic xylyl (CAX) mass tag with limited nonpolarity was selected to increase the retention of polar adducts in reversed-phase high-performance liquid chromatography (HPLC) for more detectability while maintaining resolution. We thereby detected a diversity of DNA adducts (mostly polar) by the following sequence of steps: (1) react DNA at 45°C for 2 h under aqueous conditions with CAX-B (has a benzyl bromide functional group to label active hydrogen sites) in the presence of triethylamine; (2) remove residual reagents by precipitating and washing the DNA (a convenient step); (3) digest the DNA enzymatically to nucleotides and remove unlabeled nucleotides by nonpolar solid-phase extraction (also a convenient step); and (4) detect CAX-labeled, adducted nucleotides by LC/MS 2 or a matrix-assisted laser desorption/ionization (MALDI)-MS technique., Results: Examples of the 42 DNA or RNA adducts detected, or tentatively so based on accurate mass and fragmentation data, are as follows: 8-oxo-dGMP, ethyl-dGMP, hydroxyethyl-dGMP (four isomers, all HPLC-resolved), uracil-glycol, apurinic/apyrimidinic sites, benzo[a]pyrene-dGMP, and, for the first time, benzoquinone-hydroxymethyl-dCMP. Importantly, these adducts are detected in a single procedure under a single set of conditions. Sensitivity, however, is only defined in a preliminary way, namely the latter adduct seems to be detected at a level of about 4 adducts in 10 9 nucleotides (S/N ~30)., Conclusions: CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity., (© 2021 John Wiley & Sons Ltd.)

Estragole and anethole are secondary metabolites occurring in a variety of commonly used herbs like fennel, basil, and anise. Estragole is genotoxic and carcinogenic in rodents, which depends on the formation of 1'-sulfoxyestragole after hydroxylation and subsequent sulfoconjugation catalyzed by CYP and SULT, respectively. It was hypothesized recently that anethole may be bioactivated via the same metabolic pathways. Incubating estragole with hepatic S9-fractions from rats and humans, specific adducts with hemoglobin (N-(isoestragole-3-yl)-valine, IES-Val) and DNA (isoestragole-2'-deoxyguanosine and isoestragole-2'-deoxyadenosine) were formed. An isotope-dilution technique was developed for the quantification of IES-Val after cleavage with fluorescein isothiocyanate (FITC) according to a modified Edman degradation. The same adducts, albeit at lower levels, were also detected in reactions with anethole, indicating the formation of 3'-hydroxyanethole and the reactive 3'-sulfoxyanethole. Finally, we conducted a pilot investigation in which IES-Val levels in human blood were determined during and after the consumption of an estragole- and anethole-rich fennel tea for four weeks. A significant increase of IES-Val levels was observed during the consumption phase and followed by a continuous decrease during the washout period. IES-Val may be used to monitor the internal exposure to the common reactive genotoxic metabolites of estragole and anethole, 1'-sulfoxyestragole and 3'-sulfoxyanethole, respectively., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.

Pyrrolizidine alkaloids (PAs) and PA N-oxides are common phytotoxins produced by over 6000 plant species. Humans are frequently exposed to PAs via ingestion of PA-containing herbal products or PA-contaminated foods. PAs require metabolic activation to form pyrrole-protein adducts and pyrrole-DNA adducts which lead to cytotoxicity and genotoxicity. Individual PAs differ in their metabolic activation patterns, which may cause significant difference in toxic potency of different PAs. This review discusses the current knowledge and recent advances of metabolic pathways of different PAs, especially the metabolic activation and metabolism-mediated cytotoxicity and genotoxicity, and the risk evaluation methods of PA exposure. In addition, this review provides perspectives of precision toxicity assessment strategies and biomarker development for the risk control and translational investigations of human intoxication by PAs.

We examine the use of the truncated singular value decomposition and Tikhonov regularization in standard form to address ill-posed least squares problems Ax = b that frequently arise in molecular mechanics force field parameter optimization. We illustrate these approaches by applying them to dihedral parameter optimization of genotoxic polycyclic aromatic hydrocarbon-DNA adducts that are of interest in the study of chemical carcinogenesis. Utilizing the discrete Picard condition and/or a well-defined gap in the singular value spectrum when A has a well-determined numerical rank, we are able to systematically determine truncation and in turn regularization parameters that are correspondingly used to produce truncated and regularized solutions to the ill-posed least squares problem at hand. These solutions in turn result in optimized force field dihedral terms that accurately parameterize the torsional energy landscape. As the solutions produced by this approach are unique, it has the advantage of avoiding the multiple iterations and guess and check work often required to optimize molecular mechanics force field parameters.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in environments, and some of them are causative agents of human cancer. Previous studies concluded that benzo[a]pyrene-7,8-dione (BPQ), which is one kind of carcinogenic PAH metabolites, forms covalently bonded adducts with DNA, and the major adduct formed is a deoxyguanosine adduct. In this work, we investigate the interactions between BPQ and DNA molecules via first-principles calculations. We identify six possible DNA adducts with BPQ. In addition to the four adducts forming covalent bonds, there are two adducts bound purely by van der Waals (vdW) interactions. Remarkably, the two vdW-bound adducts have comparable, if not larger, binding energies as the covalent adducts. The results may help us gain more understanding of the interactions between PAH metabolites and DNA.

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N '-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)- N -pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)- N -pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and N 6 -[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine ( N 6 -HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N 6 -[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine ( N 6 -Py-THF-dAdo) and 6-[2-(3-pyridyl)- N -pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N 6 -Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH 3 CN led to the formation of Py-Py-dN both in vitro and in vivo , which was confirmed by its isotopically labeled internal standard [pyridine- d 4 ]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA structure and are now considered invaluable resources in the goal of relating structure to function. Recently, the power of deep sequencing and careful chemical probe design have merged, permitting researchers to obtain a holistic understanding of how RNA structure can be utilized to control RNA biology transcriptome-wide. Within this review, we outline the recent advancements in chemical probe design for interrogating RNA structures inside cells and discuss the recent advances in our understanding of RNA biology through the lens of chemical probing., (© 2020 Wiley-VCH GmbH.)

Alkylation represents a main form of DNA damage. The N 2 position of guanine is frequently alkylated in DNA. The SOS-induced polymerases have been shown to be capable of bypassing various DNA damage products in Escherichia coli . Herein, we explored the influences of four N 2 -alkyl-dG lesions (alkyl = ethyl, n -butyl, isobutyl, or sec -butyl) on DNA replication in AB1157 E. coli cells and the corresponding strains with polymerases (Pol) II, IV, and V being individually or simultaneously knocked out. We found that N 2 -Et-dG is slightly less blocking to DNA replication than the N 2 -Bu-dG lesions, which display very similar replication bypass efficiencies. Additionally, Pol II and, to a lesser degree, Pol IV and Pol V are required for the efficient bypass of the N 2 -alkyl-dG adducts, and none of these lesions was mutagenic. Together, our results support that the efficient replication across small N 2 -alkyl-dG DNA adducts in E. coli depends mainly on Pol II.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B 1 (AFB 1 ) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB 1 -FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB 1 -FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB 1 -FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB 1 -FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB 1 -FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB 1 -FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Smoking is a leading cause of lung cancer, accounting for 81% of lung cancer cases. Tobacco smoke contains over 5000 compounds, of which more than 70 have been classified as human carcinogens. Of the many tobacco smoke constituents, 1,3-butadiene (BD) has a high cancer risk index due to its tumorigenic potency and its abundance in cigarette smoke. The carcinogenicity of BD has been attributed to the formation of several epoxide metabolites, of which 1,2,3,4-diepoxybutane (DEB) is the most toxic and mutagenic. DEB is formed by two oxidation reactions carried out by cytochrome P450 monooxygenases, mainly CYP2E1. Glutathione-S-transferase theta 1 (GSTT1) facilitates the conjugation of DEB to glutathione as the first step of its detoxification and subsequent elimination via the mercapturic acid pathway. Human biomonitoring studies have revealed a strong association between GSTT1 copy number and urinary concentrations of BD-mercapturic acids, suggesting that it plays an important role in the metabolism of BD. To determine the extent that GSTT1 genotype affects the susceptibility of individuals to the toxic and genotoxic properties of DEB, GSTT1 negative and GSTT1 positive HapMap lymphoblastoid cell lines were treated with DEB, and the extent of apoptosis and micronuclei (MN) formation was assessed. These toxicological end points were compared to the formation of DEB-GSH conjugates and 1,4- bis -(guan-7-yl)-2,3-butanediol ( bis -N7G-BD) DNA-DNA cross-links. GSTT1 negative cell lines were more sensitive to DEB-induced apoptosis as compared to GSTT1 positive cell lines. Consistent with the protective effect of GSH conjugation against DEB-derived apoptosis, GSTT1 positive cell lines formed significantly more DEB-GSH conjugate than GSTT1 negative cell lines. However, GSTT1 genotype did not affect formation of MN or bis -N7G-BD cross-links. These results indicate that GSTT1 genotype significantly influences BD metabolism and acute toxicity.

Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O 6 -[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O 6 -POB-G) and O 6 -methylguanine (O 6 -Me-G) adducts in DNA. These adducts can be directly repaired by O 6 -alkylguanine-DNA alkyltransferase (AGT). Repair begins by flipping the damaged base out of the DNA helix. AGT binding and base-flipping have been previously studied using pyrrolocytosine as a fluorescent probe paired to the O 6 -alkylguanine lesion, but low fluorescence yield limited the resolution of steps in the repair process. Here, we utilize the highly fluorescent 6-phenylpyrrolo-2'-deoxycytidine (6-phenylpyrrolo-C) to investigate AGT-DNA interactions. Synthetic oligodeoxynucleotide duplexes containing O 6 -POB-G and O 6 -Me-G adducts were placed within the CpG sites of codons 158, 245, and 248 of the p53 tumor suppressor gene and base-paired to 6-phenylpyrrolo-C in the opposite strand. Neighboring cytosine was either unmethylated or methylated. Stopped-flow fluorescence measurements were performed by mixing the DNA duplexes with C145A or R128G AGT variants. We observe a rapid, two-step, nearly irreversible binding of AGT to DNA followed by two slower steps, one of which is base-flipping. Placing 5-methylcytosine immediately 5' to the alkylated guanosine causes a reduction in rate constant of nucleotide flipping. O 6 -POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O 6 -Me-G at the same position. A similar effect was not observed at other codons., (© 2020 Wiley Periodicals LLC.)

Etheno (ε)-adducts, e.g., 1,N 2 -ε-guanine (1,N 2 -ε-G) and 1,N 6 -ε-adenine (1,N 6 -ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N 2 -ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N 2 -ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP. This preference for purines was also seen in the first extension step. Analysis of full-length extension products by LC-MS/MS revealed that G accounted for 85% of nucleotides inserted opposite 1,N 2 -ε-G in single base insertion, and 63% of bases inserted in the first extension step. Extension from the correct nucleotide pair (C) was not observed, but the primer with A paired opposite 1,N 2 -ε-G was readily extended. Crystal structures of ternary hpol η insertion-stage complexes with nonhydrolyzable nucleotides dAMPnPP or dCMPnPP showed a syn orientation of the adduct, with the incoming A staggered between adducted base and the 5'-adjacent T, while the incoming C and adducted base were roughly coplanar. The formation of a bifurcated H-bond between incoming dAMPnPP and 1,N 2 -ε-G and T, compared with the single H-bond formed between incoming dCMPnPP and 1,N 2 -ε-G, may account for the observed facilitated insertion of dGTP and dATP. Thus, preferential insertion of purines by hpol η across from etheno adducts contributes to distinct outcomes in error-prone DNA replication., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: DNA, the hereditary material of a human cell generally exists as Watson-Crick base paired double-stranded B-DNA. Studies suggest that DNA can also exist in non-B forms, such as four stranded G-quadruplexes (G4 DNA). Recently, our studies revealed that the regions of DNA that can fold into G-quadruplex structures are less sensitive to ionizing radiation (IR) compared to B-DNA. Importantly, we reported that the planar G-quartet of a G4 structure is shielded from radiation induced DNA breaks, while the single- and double-stranded DNA regions remained susceptible. Thus, in the present study, we investigate whether telomeric repeat DNA present at the end of telomere, known to fold into G4 DNA can protect from radiation induced damages including strand breaks, oxidation of purines and bulky adduct formation on DNA., Materials and Methods: For plasmid irradiation assay, plasmids containing human telomeric repeat DNA sequence TTAGGG (0.8 kb or 1.8 kb) were irradiated with increasing doses of IR along with appropriate control plasmids and products were resolved on 1% agarose gel. Radioprotection was evaluated based on extent of conversion of supercoiled to nicked or linear forms of the DNA following irradiation. Formation of G-quadruplex structure on supercoiled DNA was evaluated based on circular dichroism (CD) spectroscopy studies. Cleavage of radiation induced oxidative damage and extent of formation of nicks was further evaluated using base and nucleotide excision repair proteins., Results: Results from CD studies showed that the plasmid DNA harboring human telomeric repeats (TTAGGG) can fold into G-quadruplex DNA structures. Further, results showed that human telomeric repeat sequence when present on a plasmid can protect the plasmid DNA against IR induced DNA strand breaks, unlike control plasmids bearing random DNA sequence., Conclusions: Human telomeric repeat sequence when present on plasmids can fold into G-quadruplex DNA structures, and can protect the DNA against IR induced DNA strand breaks and oxidative damage. These results in conjunction with our previous studies suggest that telomeric repeat sequence imparts less sensitivity to IR and thus telomeres of chromosomes are protected from radiation.

Nitrogen mustards are among the first modern anticancer chemotherapeutics that are still widely used as non-specific anticancer alkylating agents. While the mechanism of action of mustard drugs involves the generation of DNA interstrand cross-links, the predominant lesions produced by these drugs are nitrogen half-mustard-N7-dG (NHMG) adducts. The bulky major groove lesion NHMG, if left unrepaired, can be bypassed by translesion synthesis (TLS) DNA polymerases. However, studies of the TLS past NHMG have not been reported so far. Here, we present the first synthesis of an oligonucleotide containing a site-specific NHMG. We also report kinetic and structural characterization of human DNA polymerase η (polη) bypassing NHMG. The templating NHMG slows dCTP incorporation ∼130-fold, while it increases the misincorporation frequency ∼10-30-fold, highlighting the promutagenic nature of NHMG. A crystal structure of polη incorporating dCTP opposite NHMG shows a Watson-Crick NHMG:dCTP base pair with a large propeller twist angle. The nitrogen half-mustard moiety fits snugly into an open cleft created by the Arg61-Trp64 loop of polη, suggesting a role of the Arg61-Trp64 loop in accommodating bulky major groove adducts during lesion bypass. Overall, our results presented here to provide first insights into the TLS of the major DNA adduct formed by nitrogen mustard drugs., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (K D ) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants K D of unmodified and lesion-containing G*:Del complexes, showed that the K D values were -2.5-3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 β-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes., (Copyright © 2020. Published by Elsevier B.V.)

The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC., (Copyright © 2020 Elsevier B.V. All rights reserved.)

E. coli AlkB and human ALKBH2 belong to the AlkB family enzymes, which contain several α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases that repair alkylated DNA. Specifically, the AlkB enzymes catalyze decarboxylation of α-KG to generate a high-valent Fe(IV)-oxo species that oxidizes alkyl groups on DNA adducts. AlkB and ALKBH2 have been reported to differentially repair select etheno adducts, with preferences for 1,N 6 -ethenoadenine (1,N 6 -εA) and 3,N 4 -ethenocytosine (3,N 4 -εC) over 1,N 2 -ethenoguanine (1,N 2 -εG). However, N 2 ,3-ethenoguanine (N 2 ,3-εG), the most common etheno adduct, is not repaired by the AlkB enzymes. Unfortunately, a structural understanding of the differential activity of E. coli AlkB and human ALKBH2 is lacking due to challenges acquiring atomistic details for a range of substrates using experiments. This study uses both molecular dynamics (MD) simulations and ONIOM(QM:MM) calculations to determine how the active site changes upon binding each etheno adduct and characterizes the corresponding catalytic impacts. Our data reveal that the preferred etheno substrates (1,N 6 -εA and 3,N 4 -εC) form favorable interactions with catalytic residues that situate the lesion near the Fe(IV)-oxo species and permit efficient oxidation. In contrast, although the damage remains correctly aligned with respect to the Fe(IV)-oxo moiety, repair of 1,N 2 -εG is mitigated by increased solvation of the active site and a larger distance between Fe(IV)-oxo and the aberrant carbons. Binding of non-substrate N 2 ,3-εG in the active site disrupts key DNA-enzyme interactions, and positions the aberrant carbon atoms even further from the Fe(IV)-oxo species, leading to prohibitively high barriers for oxidative catalysis. Overall, our calculations provide the first structural insight required to rationalize the experimentally-reported substrate specificities of AlkB and ALKBH2 and thereby highlight the roles of several active site residues in the repair of etheno adducts that directly correlates with available experimental data. These proposed catalytic strategies can likely be generalized to other α-KG/Fe(II)-dependent dioxygenases that play similar critical biological roles, including epigenetic and post-translational regulation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

6-Nitrochrysene (6-NC) is a potent mutagen in bacteria and carcinogenic in animals. It is the most potent carcinogen ever tested in newborn mouse assay. DNA lesions resulting from 6-NC modification are likely to induce mutations if they are not removed by cellular defense pathways prior to DNA replication. Earlier studies showed that 6-NC-derived C8-2'-deoxyadenosine adduct, N-(dA-8-yl)-6-AC, is very slowly repaired in human cells. In this study, we have investigated replication of N-(dA-8-yl)-6-AC in human embryonic kidney (HEK 293T) cells and the roles of translesion synthesis (TLS) DNA polymerases in bypassing it. Replication of a plasmid containing a single site-specific N-(dA-8-yl)-6-AC adduct in HEK 293 T cells showed that human DNA polymerase (hPol) η and hPol κ played important roles in bypassing the adduct, since TLS efficiency was reduced to 26 % in the absence of these two polymerases compared to 83 % in polymerase-competent HEK 293T cells. The progeny from HEK 293T cells provided 12.7 % mutants predominantly containing A→T transversions. Mutation frequency (MF) was increased to 17.8 % in hPol η-deficient cells, whereas it was decreased to 3.3 % and 3.9 % when the adduct containing plasmid was replicated in hPol κ- and hPol ζ-deficient cells, respectively. The greatest reduction in MF by more than 90 % (to MF 1.2 %) was observed in hPol ζ-knockout cells in which hPol κ was knocked down. Taken together, these results suggest that hPol κ and hPol ζ are involved in the error-prone TLS of N-(dA-8-yl)-6-AC, while hPol η performs error-free bypass., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cellular DNA is constantly chemically altered by exogenous and endogenous agents. As all processes of life depend on the transmission of the genetic information, multiple biological processes exist to ensure genome integrity. Chemically damaged DNA has been linked to cancer and aging, therefore it is of great interest to map DNA damage formation and repair to elucidate the distribution of damage on a genome-wide scale. While the low abundance and inability to enzymatically amplify DNA damage are obstacles to genome-wide sequencing, new developments in the last few years have enabled high-resolution mapping of damaged bases. Recently, a number of DNA damage sequencing library construction strategies coupled to new data analysis pipelines allowed the mapping of specific DNA damage formation and repair at high and single nucleotide resolution. Strikingly, these advancements revealed that the distribution of DNA damage is heavily influenced by chromatin states and the binding of transcription factors. In the last seven years, these novel approaches have revealed new genomic maps of DNA damage distribution in a variety of organisms as generated by diverse chemical and physical DNA insults; oxidative stress, chemotherapeutic drugs, environmental pollutants, and sun exposure. Preferred sequences for damage formation and repair have been elucidated, thus making it possible to identify persistent weak spots in the genome as locations predicted to be vulnerable for mutation. As such, sequencing DNA damage will have an immense impact on our ability to elucidate mechanisms of disease initiation, and to evaluate and predict the efficacy of chemotherapeutic drugs.

Translesion synthesis (TLS) through DNA adducts of antitumor platinum complexes has been an interesting aspect of DNA synthesis in cells treated with these metal-based drugs because of its correlation to drug sensitivity. We utilized model systems employing a DNA lesion derived from a site-specific monofunctional adduct formed by antitumor [PtCl(en)(L)](NO 3 ) 2 (complex AMD, en = ethane-1,2-diamine, L = N -[2-(acridin-9-ylamino)ethyl]- N -methylpropionamidine) at a unique G residue. The catalytic efficiency of TLS DNA polymerases, which differ in their processivity and fidelity for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the adduct of AMD, was investigated. For a deeper understanding of the factors that control the bypass of the site-specific adducts of AMD catalyzed by DNA polymerases, we also used microscale thermophoresis (MST) to measure the thermodynamic changes associated with TLS across a single, site-specific adduct formed in DNA by AMD. The relative catalytic efficiency of the investigated DNA polymerases for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the AMD adduct, was reduced. Nevertheless, incorporation of the correct C opposite the G modified by AMD of the template strand was promoted by an increasing thermodynamic stability of the resulting duplex. The reduced relative efficiency of the investigated DNA polymerases may be a consequence of the DNA intercalation of the acridine moiety of AMD and the size of the adduct. The products of the bypass of this monofunctional lesion produced by AMD and DNA polymerases also resulted from the misincorporation of dNTPs opposite the platinated G residues. The MST analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dNTPs by DNA polymerases.

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.

Exposure to aristolochic acid I and II (AAI and AAII) has been implicated in aristolochic acid nephropathy and urothelial carcinoma. The toxicological effects of AAs are attributed to their ability to form aristolacatam (AL)-purine DNA adducts. Among these lesions, the AL-adenine (ALI-N 6 -A and ALII-N 6 -A) adducts cause the "signature" A → T transversion mutations associated with AA genotoxicity. To provide the currently missing structural basis for the induction of these signature mutations, the present work uses classical all-atom molecular dynamics simulations to examine different (i.e., preinsertion, insertion, and postextension) stages of replication past the most abundant AA adduct (ALI-N 6 -A) by a representative lesion-bypass DNA polymerase (Dpo4). Our analysis reveals that, before dNTP incorporation (i.e., preinsertion step), ALI-N 6 -A adopts a nearly planar conformation at the N 6 -linkage and the ALI moiety intercalates within the DNA helix. Since this conformation occupies the dNTP binding site, the same planar lesion conformation results in a significant distortion of the polymerase active site at the insertion step and therefore replication will likely not be successful. However, if ALI-N 6 -A undergoes a small conformational change to introduce non-planarity at the N 6 -linkage during the insertion step, minimal distortion occurs in the Dpo4 active site upon incorporation of dATP. This insertion and subsequent extension would initially lead to A:A mismatches and then result in A → T transversion mutations during the second round of replication. In contrast, if a large conformation flip of the ALI moiety occurs at the insertion step to reorient the bulky moiety from an intercalated position into the major groove, dTTP (non-mutagenic) incorporation will be favored. Molecular dynamics (MD) simulations on postextension complexes reveal that damaged DNA will likely further rearrange during later replication steps to acquire a base-displaced intercalated conformation that is similar to that previously reported for (unbound) ALI-N 6 -A adducted DNA, with the exception of slight non-planarity at the lesion site. Overall, our results provide a structural explanation for both the successful non-mutagenic lesion bypass and the preferential misincorporation of dATP opposite ALI-N 6 -A and thereby rationalize the previously reported induction of A → T signature transversion mutations associated with AAs. This work should thereby inspire future biochemical experiments and modeling studies on the replication of this important class of DNA lesions by related human translesion synthesis polymerases.

Proteins that act on DNA, or are in close proximity to it, can become inadvertently crosslinked to DNA and form highly toxic lesions, known as DNA-protein crosslinks (DPCs). DPCs are generated by different chemotherapeutics, environmental or endogenous sources of crosslinking agents, or by lesions on DNA that stall the catalytic cycle of certain DNA processing enzymes. These bulky adducts impair processes on DNA such as DNA replication or transcription, and therefore pose a serious threat to genome integrity. The large diversity of DPCs suggests that there is more than one canonical mechanism to repair them. Indeed, many different enzymes have been shown to act on DPCs by either processing the protein, the DNA or the crosslink itself. In addition, the cell cycle stage or cell type are likely to dictate pathway choice. In recent years, a detailed understanding of DPC repair during S phase has started to emerge. Here, we review the current knowledge on the mechanisms of replication-coupled DPC repair, and describe and also speculate on possible pathways that remove DPCs outside of S phase. Moreover, we highlight a recent paradigm shifting finding that indicates that DPCs are not always detrimental, but can also play a protective role, preserving the genome from more deleterious forms of DNA damage., (Copyright © 2020. Published by Elsevier B.V.)

The clinical efficiency of Pt(II)-based drugs is founded on articulate mechanisms of action. Indeed it depends on a balanced combination of metal ion reactivity towards proteins and nucleic acids. Here we analysed the effect of two trans-platinum planar amines in comparison to cisplatin and transplatin on the DNA processivity by human topoisomerases I and IIα. Each tested metal complex produces DNA adducts with unique geometrical features and, consistently, they exert different effects on the activity of tested enzymes. Moreover, our results highlighted more subtle consequences on the enzymatic activity by the tested metal complexes which derive from a combination of preferential DNA or protein platination. Moreover, we observed that it is not possible to predict the overall output based only on the cis- vs trans- geometry of the tested metal complexes. This variable behaviour reflects the chemical reactivity profile of each single metal complex and can be usefully addressed to describe their different properties in the complex physiological environment., Competing Interests: Declaration of competing interest The authors have nothing to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

DNA topoisomerases alleviate the torsional stress that is generated by processes that are central to genome metabolism such as transcription and DNA replication. To do so, these enzymes generate an enzyme intermediate known as the cleavage complex in which the topoisomerase is covalently linked to the termini of a DNA single- or double-strand break. Whilst cleavage complexes are normally transient they can occasionally become abortive, creating protein-linked DNA breaks that threaten genome stability and cell survival; a process promoted and exploited in the cancer clinic by the use of topoisomerase 'poisons'. Here, we review the consequences to genome stability and human health of abortive topoisomerase-induced DNA breakage and the cellular pathways that cells have adopted to mitigate them, with particular focus on an important class of enzymes known as tyrosyl-DNA phosphodiesterases., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Topoisomerases play a pivotal role in ensuring DNA metabolisms during replication, transcription and chromosomal segregation. To manage DNA topology, topoisomerases generate break(s) in the DNA backbone by forming transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and topoisomerase catalytic tyrosyl residues. Topoisomerases have been identified as the cellular targets of a variety of anti-cancer drugs (e.g. topotecan, irinotecan, etoposide and doxorubicin, and antibiotics (e.g. ciprofloxacin and levofloxacin). These drugs, as well as other exogenous and endogenous agents, convert the transient TOPcc into persistent TOPcc, which we refer to as topoisomerase DNA-protein crosslinks (TOP-DPC) that challenge genome integrity and lead to cell death if left unrepaired. Proteolysis of the bulky protein component of TOP-DPC (debulking) is a poorly understood repair process employed across eukaryotes. TOP-DPC proteolysis can be achieved either by the ubiquitin-proteasome pathway (UPP) or by non-proteasomal proteases, which are typified by the metalloprotease SPRTN/WSS1. Debulking of TOP-DPC exposes the phosphotyrosyl bonds, hence enables tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) to access and cleave the bonds. In this review, we focus on current knowledge of the protease pathways for debulking TOP-DPC and highlighting recent advances in understanding the mechanisms regulating the proteolytic repair pathways. We also discuss the avenues that are being exploited to target the proteolytic repair pathways for improving the clinical outcome of topoisomerase inhibitors., (Published by Elsevier B.V.)

The formation and repair of N 2 -( trans -isosafrol-3'-yl)-2'-deoxyguanosine (S-3'- N 2 -dG) DNA adduct derived from the spice and herbal alkenylbenzene constituent safrole were investigated. DNA adduct formation and repair were studied in vitro and using molecular dynamics (MD) simulations. DNA adduct formation was quantified using liquid chromatography-mass spectrometry (LCMS) in wild type and NER (nucleotide excision repair) deficient CHO cells and also in HepaRG cells and primary rat hepatocytes after different periods of repair following exposure to safrole or 1'-hydroxysafrole (1'-OH safrole). The slower repair of the DNA adducts found in NER deficient cells compared to that in CHO wild type cells indicates a role for NER in repair of S-3'- N 2 -dG DNA adducts. However, DNA repair in liver cell models appeared to be limited, with over 90% of the adducts remaining even after 24 or 48 h recovery. In our further studies, MD simulations indicated that S-3'- N 2 -dG adduct formation causes only subtle changes in the DNA structure, potentially explaining inefficient activation of NER. Inefficiency of NER mediated repair of S-3'- N 2 -dG adducts points at persistence and potential bioaccumulation of safrole DNA adducts upon daily dietary exposure.

The instability of electrophilic reactive metabolites in in vitro metabolism studies makes their accurate analysis challenging. To stabilise the reactive compounds prior to their analysis, different trapping agents, such as thiols, amines and cob(I)alamin, have earlier been tested depending on the metabolites to be analysed and the type of study. In the present work, DNA is introduced as a trapping agent for measuring the formation of bulky electrophilic metabolites. Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), was used as a model compound in a rat liver S9 metabolic system. Under physiological incubation conditions, B[a]P metabolises to diol epoxide (BPDE) metabolites which were trapped by DNA resulting in the formation of covalently bound DNA adducts. The methodology for analysis of these adducts included extraction of the DNA from the metabolic system, digestion of the DNA to yield nucleosides and analysis of the BPDE-adduct to deoxyguanosine (BPDE-dG) by liquid chromatography coupled to high resolution mass spectrometry (HRMS). The chromatographic conditions in combination with the high mass accuracy data (±3 ppm) was useful in resolving BPDE-dG in its protonated form from the complex set of ions present in the metabolic matrix. The method was validated in terms of sensitivity, specificity, accuracy, precision and recovery, and applied to provide a preliminary estimate of BPDE-dG levels from the metabolism of B[a]P in rat S9. The use of DNA as a trapping agent for in vitro metabolites has a potential to aid in cancer risk assessment procedure of PAHs, for instance, in inter-species comparison of metabolism to reactive metabolites and can be adapted for screening of genotoxic metabolites, e.g., from emerging environmental contaminants., Competing Interests: Declaration of Competing Interest The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author. Published by Elsevier B.V. All rights reserved.)

Background: Polycyclic aromatic hydrocarbons (PAHs) are a kind of endocrine disruptors, which can enter human body by the inhalation of PAH-containing matter and the ingestion of PAH-containing foodstuffs. Studies showed that PAHs can cross the placental barrier and might cause adverse effects on the fetus., Objectives: This meta-analysis aimed to estimate the associations between prenatal exposure to PAHs and birth weight., Methods: Articles published in English until May 8, 2020 and reported the effects of prenatal exposure to PAHs on birth weight were searched in multiple electronic databases including PubMed, the Web of Science, EMBASE and the Cochrane Library. The included studies were divided into three groups in accordance with the measurement of PAHs exposure. Then coefficient was extracted, conversed and synthesized by random-effects meta-analysis. And risk of bias was assessed for each study., Results: A total of 3488 citations were searched and only 11 studies were included finally after double assessment. We found that there were no association between PAH-DNA adducts in cord blood (low/high) (OR: 1.0, 95%CI: 0.97, 1.03), 1-hydroxy pyrene (1-HP) concentration in maternal urine (OR: 1.0, 95%CI: 0.97, 1.03) and prenatal maternal airborne PAHs exposure (OR: 0.97, 95%CI: 0.93, 1.01) and birth weight. However, we observed ethnicity may change the effects of PAHs exposure on birth weight., Conclusions: There is no significant relationship between prenatal exposure to PAHs and birth weight in our meta-analysis. Further studies are still needed for determining the effects of prenatal PAHs exposure on birth weight., Competing Interests: The authors have declared that no competing interests exist.

Topoisomerase I (TOP1) resolves DNA topology during replication and transcription. The enzyme forms an intermediate TOP1 cleavage complex (TOP1cc) through transient TOP1-DNA-protein crosslinks. Camptothecin is a frontline anticancer agent that freezes this reaction intermediate, leading to the generation of irreversible TOP1ccs that act as 3'-blocking lesions. It is widely accepted that TOP1cc is repaired via a two-step pathway involving proteasomal degradation of TOP1cc to the crosslinked peptide, followed by removal of the TOP1cc-derived peptide from DNA by tyrosyl-DNA phosphodiesterase 1 (TDP1). In the present study, we developed an assay system to estimate repair kinetics of TOP1cc separately in the first and second steps, using monoclonal antibodies against the TOP1 protein and the TOP1 catalytic site peptide-DNA complex, respectively. Although TDP1-deficient (TDP1 -/- ) TK6 cells had normal kinetics of the first step, a delay in the kinetics of the second step was observed relative to that in wild-type cells. Tyrosyl-DNA phosphodiesterase 2 (TDP2) reportedly promotes the repair of TOP1-induced DNA damage in the absence of TDP1. The present assays additionally demonstrated that TDP2 promotes the second, but not the first, step of TOP1cc repair in the absence of TDP1. We also analyzed sensitivities of TK6 cells with deficiencies in TDP1 and/or TDP2 to agents that produce 3' -blocking lesions. These experiments showed that TDP1 -/- TDP2 -/- cells were more sensitive to the agents Azidothymidine (zidovudine), Cytarabine, Abacavir, Gemcitabine, and Trifluridine than TDP1 -/- or TDP2 -/- cells. Taken together, our findings confirm the roles of TDP2 in the repair of 3'-blocking lesions., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As one of the most widespread environmental pollutants, benzo[α]pyrene is metabolized to diol epoxides and then covalently breaks the initial DNA base pairs, which has been closely related to the occurrence and development of many human cancers. High fidelity DNA polymerases play an extremely important role in maintaining the reliability or fidelity of nucleic acid replication, which is generally blocked by BP adducts. To reveal the blocking mechanism of BP, two comparative molecular dynamics simulations were performed for the thermophilic Bacillus stearothermophilus DNA polymerase I large fragment (BF) complexes with normal and BP-bound DNA duplexes. The results of global conformational changes and molecular interactions show that the association of BP leads to the rearrangement of intramolecular hydrogen bonds, impairing the molecular recognition between the polymerase and the DNA duplex. It is also found that the conformation of DNA duplex is distorted, accompanied by an increase in molecular overall rigidity. In terms of possible blocking mechanisms, the BP moiety perfectly integrates itself into the base-paired environment in a special vertical conformation and occupies the space required for the incoming nucleotide. This work provides useful dynamics and structural information for understanding the toxic effect of BP on DNA replication at atomic level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The monoalkylated N7-guanine (NM-G) adduct and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened N 5 -substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM-Fapy-G adducts in liver, lung, and spleen over 168 h. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three-times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 h post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing in vivo . The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.