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7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

DNA is continuously exposed to a variety of harmful factors, which, on the one hand, can force undesirable processes such as ageing, carcinogenesis and mutagenesis, while on the other hand, can accelerate evolutionary changes. Of all the canonical nucleosides, 2'-deoxyguanosine (dG) exhibits the lowest ionization potential, making it particularly prone to the one-electron oxidizing process. The most abundant type of nucleobase damage is constituted by 7,8-dihydro-8-oxo-2'-deoxyguanosine ( OXO dG), with an oxidation potential that is 0.56 V lower than that of canonical dG. All this has led to OXO dG, as an isolated lesion, being perceived as a sink for radical cations in the genome. In this paper, a comparative analysis of the electronic properties of an OXO GC base pair within the context of a clustered DNA lesion (CDL) has been conducted. It is based on previous DFT studies that were carried out at the M06-2x/6-31++G** level of theory in non-equilibrated and equilibrated condensed phases. The results of the comparative analysis presented here reveal the following: (A) The ionization potentials of OXO G 4 C 2 were largely unaffected by a second lesion. (B) The positive charge and spin were found predominantly on the OXO G 4 C 2 moiety. (C) The electron-hole transfers A 3 T 3 →G 4 C 2 and G 4 C 2 ←A 5 T 1 were found in the Marcus inverted region and were resistant to the presence of a second DNA lesion in close proximity. It can therefore be reasonably postulated that OXO GC becomes the sink for a radical cation migrating through the double helix, irrespective of the presence of other 2'-deoxyguanosine lesions in the CDL structure.

The genome-the source of life and platform of evolution-is continuously exposed to harmful factors, both extra- and intra-cellular. Their activity causes different types of DNA damage, with approximately 80 different types of lesions having been identified so far. In this paper, the influence of a clustered DNA damage site containing imidazolone (Iz) or oxazolone (Oz) and 7,8-dihydro-8-oxo-2'-deoxyguanosine ( OXO dG) on the charge transfer through the double helix as well as their electronic properties were investigated. To this end, the structures of oligo-Iz , d[A 1 Iz 2 A 3 OXO G 4 A 5 ]*d[T 5 C 4 T 3 C 2 T 1 ], and oligo-Oz , d[A 1 Oz 2 A 3 OXO G 4 A 5 ]*d[T 5 C 4 T 3 C 2 T 1 ], were optimized at the M06-2X/6-D95**//M06-2X/sto-3G level of theory in the aqueous phase using the ONIOM methodology; all the discussed energies were obtained at the M06-2X/6-31++G** level of theory. The non-equilibrated and equilibrated solvent-solute interactions were taken into consideration. The following results were found: (A) In all the discussed cases, OXO dG showed a higher predisposition to radical cation formation, and B) the excess electron migration toward Iz and Oz was preferred. However, in the case of oligo-Oz , the electron transfer from Oz 2 to complementary C 4 was noted during vertical to adiabatic anion relaxation, while for oligo-Iz , it was settled exclusively on the Iz 2 moiety. The above was reflected in the charge transfer rate constant, vertical/adiabatic ionization potential, and electron affinity energy values, as well as the charge and spin distribution. It can be postulated that imidazolone moiety formation within the CDL ds-oligo structure and its conversion to oxazolone can significantly influence the charge migration process, depending on the C2 carbon hybridization sp 2 or sp 3 . The above can confuse the single DNA damage recognition and removal processes, cause an increase in mutagenesis, and harm the effectiveness of anticancer therapy.

N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy•dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OxodGuo). Fapy•dG preferentially gives rise to G → T transversions and G → A transitions. However, the molecular basis by which Fapy•dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase β (Pol β), a model mammalian polymerase, bypasses a templating Fapy•dG, inserts Fapy•dGTP, and extends from Fapy•dG at the primer terminus. When Fapy•dG is present in the template, Pol β incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy•dGTP is a poor substrate but is incorporated ∼3-times more efficiently opposite dA than dC. Extension from Fapy•dG at the 3'-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy•dG is likely to be the source of the mutagenic effects of the lesion and not Fapy•dGTP. These experiments increase our understanding of the promutagenic effects of Fapy•dG., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo , but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPC MdG ), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPC MdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPC MdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPC MdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

DNA is constantly damaged by various external and internal factors. In particular, oxidative damage occurs in a steady state, and 8-oxo-2'-deoxyguanosine (oxodG) is known as the main oxidative damage. OxodG is a strong genotoxic nucleoside and is thought to be involved in the pathogenesis of cancer and neurological diseases. However, a breakthrough method to detect the position of oxodG in DNA has not yet been developed. Therefore, we attempted to develop a novel method to detect oxodG in DNA using artificial nucleosides. Recently, we have succeeded in the recognition of oxodG in DNA by a single nucleotide elongation reaction using nucleoside derivatives based on a purine skeleton with a 1,3-diazaphenoxazine unit. In this study, we developed a new nucleoside derivative with a pyrimidine skeleton in order to further improve the recognition ability and enzymatic reaction efficiency. We, therefore, designed and synthesized 2'-deoxycytidine-1,3-diazaphenoxazine (Cdap) and its triphosphate derivatives. The results showed that it was incorporated into the primer strand relative to the dG template because of its cytidine skeleton, but it was more effective at the complementary position of the oxodG template. These results indicate that the new nucleoside derivative can be considered as one of the new candidates for the detection of oxodG in DNA.

The genotoxic 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M 1 dG) DNA lesion arises from endogenous exposures to base propenals generated by oxidative damage and from exposures to malondialdehyde (MDA), produced by lipid peroxidation. Once formed, M 1 dG may oxidize, in vivo , to 3-(2-deoxy-β-D-erythropentofuranosyl)-pyrimido[1,2- f ]purine-6,10(3 H ,5 H )-dione (6-oxo-M 1 dG). The latter blocks DNA replication and is a substrate for error-prone mutagenic bypass by the Y-family DNA polymerase hpol η. To examine structural consequences of 6-oxo-M 1 dG damage in DNA, we conducted NMR studies of 6-oxo-M 1 dG incorporated site-specifically into 5' -d(C 1 A 2 T 3X4 A 5 T 6 G 7 A 8 C 9 G 10 C 11 T 12 )-3':5'-d(A 13 G 14 C 15 G 16 T 17 C 18 A 19 T 20 C 21 A 22 T 23 G 24 )-3' ( X = 6-oxo-M 1 dG). NMR spectra afforded detailed resonance assignments. Chemical shift analyses revealed that nucleobase C 21 , complementary to 6-oxo-M 1 dG, was deshielded compared with the unmodified duplex. Sequential NOEs between 6-oxo-M 1 dG and A 5 were disrupted, as well as NOEs between T 20 and C 21 in the complementary strand. The structure of the 6-oxo-M 1 dG modified DNA duplex was refined by using molecular dynamics (rMD) calculations restrained by NOE data. It revealed that 6-oxo-M 1 dG intercalated into the duplex and remained in the anti -conformation about the glycosyl bond. The complementary cytosine C 21 extruded into the major groove, accommodating the intercalated 6-oxo-M 1 dG. The 6-oxo-M 1 dG H7 and H8 protons faced toward the major groove, while the 6-oxo-M 1 dG imidazole proton H2 faced into the major groove. Structural perturbations to dsDNA were limited to the 6-oxo-M 1 dG damaged base pair and the flanking T 3 :A 22 and A 5 :T 20 base pairs. Both neighboring base pairs remained within the Watson-Crick hydrogen bonding contact. The 6-oxo-M 1 dG did not stack well with the 5'-neighboring base pair T 3 :A 22 but showed improved stacking with the 3'-neighboring base pair A 5 :T 20 . Overall, the base-displaced intercalated structure was consistent with thermal destabilization of the 6-oxo-M 1 dG damaged DNA duplex; thermal melting temperature data showed a 15 °C decrease in T m compared to the unmodified duplex. The structural consequences of 6-oxo-M 1 dG formation in DNA are evaluated in the context of the chemical biology of this lesion.

Sequence context influences structural characteristics and repair of DNA adducts, but there is limited information on how epigenetic modulation affects conformational heterogeneity and bypass of DNA lesions. Lesions derived from the environmental pollutant 2-nitrofluorene have been extensively studied as chemical carcinogenesis models; they adopt a sequence-dependent mix of two significant conformers: major groove binding (B) and base-displaced stacked (S). We report a conformation-dependent bypass of the N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (dG-FAF) lesion in epigenetic sequence contexts (d[5'-CTTCTC#G*NCCTCATTC-3'], where C# is C or 5-methylcytosine (5mC), G* is G or G-FAF, and N is A, T, C or G). FAF-modified sequences with a 3' flanking pyrimidine were better bypassed when the 5' base was 5mC, whereas sequences with a 3' purine exhibited the opposite effect. The conformational basis behind these variations differed; for -CG*C- and -CG*T-, bypass appeared to be inversely correlated with population of the duplex-destabilizing S conformer. On the other hand, the connection between conformation and a decrease in bypass for flanking purines in the 5mC sequences relative to C was more complex. It could be related to the emergence of a disruptive non-S/B conformation. The present work provides novel conformational insight into how 5mC influences the bypass efficiency of bulky DNA damage., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The development and establishment of oral squamous cell carcinoma are confined to carcinogenesis, which involves oxidative stress via oxygen-free radical production as a hydroxyl radical (HO•), considered the most important cause of oxidative damage to basic biomolecules since it targets DNA strands. 8-Hydroxy-2´-deoxyguanosine (8-OHdG) is considered a free radical with a promutagenic capacity due to its ability to pair with adenosine instead of cytosine during replication., Material and Methods: We collected 30 paraffin-embedded tissue samples of OSCC from patients treated between 2013 and 2018. We recorded risk habits, disease stage, disease free survival and death with at least 3 years of follow-up. 8-Hydroxyguanosine was evaluated by immunohistochemistry and subsequently classified as weak-moderate or strong positive expression. Additionally, we noted whether it was expressed in the cytoplasm and/or nucleus., Results: Most of the cases expressed 8-OHdG with a strong intensity (80%). All neoplastic cells were preferentially stained in only the cytoplasm (70.0%), but nuclear positivity was found in 30%, independent of the intensity. Based on the location in the cytoplasm and/or nucleus, tumors >4 cm showed a high frequency (95.5%) of 8-OHdG expression in only the cytoplasm, with a significant difference (p value 0.001). Additionally, overall survival was affected when immunoexpression was present in the cytoplasm and nucleus because all deaths were in this group were statistically significant (p value = 0.001)., Conclusions: All tumors showed DNA oxidative damage, and 8-OHdG was preferentially expressed in the cytoplasm. This finding was associated with tumor size and, when present in the nucleus, might also be related to death.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPDQ), one of the oxidation products of rubber antioxidant 6PPD, has been identified as a novel toxicant to many organisms. However, an understanding of its underlying toxicity mechanisms remained elusive. In this study, we reported that 6PPDQ could react with deoxyguanosine to form one isomer of 3-hydroxy-1, N 2 -6PPD-etheno-2'-deoxyguanosine (6PPDQ-dG). Next, by employing an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method, we found that 6PPDQ-dG could be detected in genomic DNA from 6PPDQ-treated mammalian cells and Chlamydomonas reinhardtii. We observed positive correlations between concentrations of exogenous 6PPDQ and the amounts of 6PPDQ-dG, and a recovery period after removal of 6PPDQ also led to decreased levels of the adduct in both organisms, which suggested potential repair pathways for this adduct in mammalian cells and unicellular algae. Additionally, we extracted the genomic DNA from tissues of frozen capelin and observed substantial amounts of the adduct in roe and gills, as well as livers at a relatively lower level. These results provided insights into the target organs and tissues that 6PPDQ might accumulate or harm fish. Overall, our study provides a new understanding of the mechanisms of toxicity of 6PPDQ in mammalian cells and aqueous organisms., 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 © 2023 Elsevier B.V. All rights reserved.)

Every 24 h, roughly 3 × 10 17 incidences of DNA damage are generated in the human body as a result of intra- or extra-cellular factors. The structure of the formed lesions is identical to that formed during radio- or chemotherapy. Increases in the clustered DNA damage (CDL) level during anticancer treatment have been observed compared to those found in untreated normal tissues. 7,8-dihydro-8-oxo-2'-deoxyguanosine ( OXO G) has been recognized as the most common lesion. In these studies, the influence of OXO G, as an isolated (oligo- O G) or clustered DNA lesion (oligo- O G O G), on charge transfer has been analyzed in comparison to native oligo-G. DNA lesion repair depends on the damage recognition step, probably via charge transfer. Here the electronic properties of short ds-oligonucleotides were calculated and analyzed at the M062x/6-31++G** level of theory in a non-equilibrated and equilibrated solvent state. The rate constant of hole and electron transfer according to Marcus' theory was also discussed. These studies elucidated that OXO G constitutes the sink for migrated radical cations. However, in the case of oligo- O G O G containing a 5'- OXO GA XOX G-3' sequence, the 3'-End OXO G becomes predisposed to electron-hole accumulation contrary to the undamaged GAG fragment. Moreover, it was found that the 5'-End OXO G present in an OXO GA OXO G fragment adopts a higher adiabatic ionization potential than the 2'-deoxyguanosine of an undamaged analog if both ds-oligos are present in a cationic form. Because increases in CDL formation have been observed during radio- or chemotherapy, understanding their role in the above processes can be crucial for the efficiency and safety of medical cancer treatment.

Genetic information is continuously exposed to harmful factors, both intra- and extracellular. Their activity can lead to the formation of different types of DNA damage. Clustered lesions (CDL) are problematic for DNA repair systems. In this study, the short ds-oligos with a CDL containing ( R ) or ( S ) 2Ih and OXO G in their structure were chosen as the most frequent in vitro lesions. In the condensed phase, the spatial structure was optimized at the M062x/D95**:M026x/sto-3G level of theory, while the electronic properties were optimized at the M062x/6-31++G** level. The influence of equilibrated and non-equilibrated solvent-solute interactions was then discussed. It was found that the presence of ( R )2Ih in the ds-oligo structure causes a greater increase in structure sensitivity towards charge adoption than ( S )2Ih, while OXO G shows high stability. Moreover, the analysis of charge and spin distribution reveals the different effects of 2Ih diastereomers. Additionally, the adiabatic ionization potential was found as follows for ( R )-2Ih and ( S )-2Ih in eV: 7.02 and 6.94. This was in good agreement with the AIP of the investigated ds-oligos. It was found that the presence of ( R )-2Ih has a negative influence on excess electron migration through ds-DNA. Finally, according to the Marcus theory, the charge transfer constant was calculated. The results presented in the article show that both diastereomers of 5-carboxamido-5-formamido-2-iminohydantoin should play a significant role in the CDL recognition process via electron transfer. Moreover, it should be pointed out that even though the cellular level of ( R and S )-2Ih has been obscured, their mutagenic potential should be at the same level as other similar guanine lesions found in different cancer cells.

Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven N 2 -dG lesions of different steric bulkiness. In the presence of Mg 2+ ions, bulky N 2 -isobutyl-dG, N 2 -benzyl-dG, N 2 -methyl(1-naphthyl)-dG, N 2 -methyl(9-anthracenyl)-dG, N 2 -methyl(1-pyrenyl)-dG, and N 2 -methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite N 2 -ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn 2+ ions. When Mn 2+ replaced Mg 2+ , PrimPol carried out DNA synthesis on all DNA templates with N 2 -dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion "skipping" mechanism. The TLS activity of PrimPol opposite N 2 -ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1'-C1' distances for the N 2 -isobutyl-dG and N 2 -benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.

Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, N 7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.

Oxidative lesions, such as 8-oxo-dG and 8-oxo-dA, are continuously generated from exposure to reactive oxygen species. While 8-oxo-dG has been extensively studied, 8-oxo-dA has not received as much attention until recently. Herein, we report the synthesis of duplex DNAs incorporating dA, 8-oxo-dA, 7-deaza-dA, 8-Br-dA, and 8-Br-7-deaza-dA, which have different substitutions at 7- and 8-position, for the investigation into the implications of N7-hydrogen and C8-keto on the base pairing preference, mutagenic potential and repair of 8-oxo-dA. Base pairing study suggested that the polar N7-hydrogen and C8-keto of 8-oxo-dA, rather than the syn-preference, might be essential for 8-oxo-dA to form a stable base pair with dG. Insertion and extension studies using KF-exo - and human DNA polymerase β indicated that the efficient dGTP insertion opposite 8-oxo-dA and extension past 8-oxo-dA:dG are contingent upon not only the stable base pair with dG, but also the flexibility of the active site in polymerase. The N7-hydrogen in 8-oxo-dA or C7-hydrogen in 7-deaza-dA and 8-Br-7-deaza-dA was suggested to be important for the recognition by hOGG1, although the excision efficiencies of 7-deaza-dA and 8-Br-7-deaza-dA were much lower than 8-oxo-dA. This study provides an insight into the structure-function relationship of 8-oxo-dA by nucleotide analogues., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Epidemiological and mechanistic studies suggest that processed and red meat consumption and tobacco smoking are associated with colorectal cancer (CRC) risk. Several classes of carcinogens, including N -nitroso compounds (NOCs) in processed meats and heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled meats and tobacco smoke, undergo metabolism to reactive intermediates that may form mutation-inducing DNA adducts in the colorectum. Heme iron in red meat may contribute to oxidative DNA damage and endogenous NOC formation. However, the chemicals involved in colorectal DNA damage and the paradigms of CRC etiology remain unproven. There is a critical need to establish physicochemical methods for identifying and quantitating DNA damage induced by genotoxicants in the human colorectum. We established robust nano-liquid chromatography/high-resolution accurate mass Orbitrap tandem mass spectrometry (LC/HRAMS 2 ) methods to measure DNA adducts of nine meat and tobacco-associated carcinogens and lipid peroxidation products in the liver, colon, and rectum of carcinogen-treated rats employing fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues. Some NOCs form O 6 -carboxymethyl-2'-deoxyguanosine, O 6 -methyl-2'-deoxyguanosine, and unstable quaternary N -linked purine/pyrimidine adducts, which generate apurinic/apyrimidinic (AP) sites. AP sites were quantitated following derivatization with O -(pyridin-3-yl-methyl)hydroxylamine. DNA adduct quantitation was conducted with stable isotope-labeled internal standards, and method performance was validated for accuracy and reproducibility. Limits of quantitation ranged from 0.1 to 1.1 adducts per 10 8 bases using 3 μg of DNA. Adduct formation in animals ranged from ∼1 in 10 8 to ∼1 in 10 5 bases, occurring at comparable levels in fresh-frozen and FFPE specimens for most adducts. AP sites increased by 25- to 75-fold in the colorectum and liver, respectively. Endogenous lipid peroxide-derived 3-(2-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3 H )-one (M 1 dG) and 6-oxo-M 1 dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS 2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M 1 dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M 1 dG in genomic DNA is enzymatically oxidized to 6-oxo-M 1 dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M 1 dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M 1 dG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M 1 dG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-M 1 dG blocks further replication by the enzyme.

To cope with unrepaired DNA lesions, cells are equipped with DNA damage tolerance mechanisms, including translesion synthesis (TLS). While TLS polymerases are well documented in facilitating replication across damaged DNA templates, it remains unknown whether TLS polymerases participate in transcriptional bypass of DNA lesions in cells. Herein, we employed the competitive transcription and adduct bypass assay to examine the efficiencies and fidelities of transcription across N 2 -alkyl-2'-deoxyguanosine ( N 2 -alkyl-dG, alkyl = methyl, ethyl, n -propyl, or n -butyl) lesions in HEK293T cells. We found that N 2 -alkyl-dG lesions strongly blocked transcription and elicited CC → AA tandem mutations in nascent transcripts, where adenosines were misincorporated opposite the lesions and their adjacent 5' nucleoside. Additionally, genetic ablation of Pol η, but not Pol κ, Pol ι, or Pol ζ, conferred marked diminutions in the transcriptional bypass efficiencies of the N 2 -alkyl-dG lesions, which is exacerbated by codepletion of Rev1 in Pol η-deficient background. We also observed that the repair of N 2 - n Bu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. Hence, our results provided insights into transcriptional perturbations induced by N 2 -alkyl-dG lesions and expanded the biological functions of TLS DNA polymerases.

Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin- N 4-yl radical (dC·) plays a role in DNA damage mediated by one electron oxidants, such as HOCl and ionizing radiation. However, the reactivity of dC· is not well understood. To reduce this knowledge gap, we photochemically generated dC· from a nitrophenyl oxime nucleoside and within chemically synthesized oligonucleotides from the same precursor. dC· formation is confirmed by transient UV-absorption spectroscopy in laser flash photolysis (LFP) experiments. LFP and duplex DNA cleavage experiments indicate that dC· oxidizes dG. Transient formation of the dG radical cation (dG +• ) is observed in LFP experiments. Oxidation of the opposing dG in DNA results in hole transfer when the opposing dG is part of a dGGG sequence. The sequence dependence is attributed to a competition between rapid proton transfer from dG +• to the opposing dC anion formed and hole transfer. Enhanced hole transfer when less acidic O 6-methyl-2'-deoxyguanosine is opposite dC· supports this proposal. dC· produces tandem lesions in sequences containing thymidine at the 5'-position by abstracting a hydrogen atom from the thymine methyl group. The corresponding thymidine peroxyl radical completes tandem lesion formation by reacting with the 5'-adjacent nucleotide. As dC· is reduced to dC, its role in the process is traceless and is only detectable because of the ability to independently generate it from a stable precursor. These experiments reveal that dC· oxidizes neighboring nucleotides, resulting in deleterious tandem lesions and hole transfer in appropriate sequences.

Recent advances in G-quadruplex (GQ) studies have provided evidence for their important role in key biological processes (replication, transcription, genome stability, and epigenetics). These findings imply highly specific interactions between GQ structures and cellular proteins. The details of the interaction between GQs and cellular proteins remain unknown. It is now accepted that GQ loop elements play a major role in protein recognition. It remains unclear whether and to what extent the GQ core contributes to maintaining the recognition interface. In the current paper, we used the thrombin binding aptamer as a model to study the effect of modification in the quadruplex core on the ability of aptamer to interact with thrombin. We used alpha-2'-deoxyguanosine and 8-bromo-2'-deoxyguanosine to reconfigure the core or to affect syn - anti preferences of selected dG-residues. Our data suggest that core guanines not only support a particular type of GQ architecture, but also set structural parameters that make GQ protein recognition sensitive to quadruplex topology.

The O 6 -alkylguanosine adduct O 6 -carboxymethyldeoxyguanosine ( O 6 -CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of O 6 -CMdG as well as the corresponding methyl adduct O 6 -methyldeoxyguanosine ( O 6 -MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize O 6 -CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of O 6 -CMdG and O 6 -MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an O 6 -serine-CMdG ( O 6 -Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O 6 -Ser-CMdG forms as an intermediate that spontaneously decomposes to form O 6 -CMdG. Finally, we quantified levels of O 6 -CMdG and O 6 -MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.

The seed of life is concealed in the base sequence in DNA. This macromolecule is continuously exposed to harmful factors which can cause it damage. The stability of genetic information depends on the protein efficiency of repair systems. Glycosylases are the scouts which recognize and remove damaged bases. Their efficiency depends on how rapidly they recognize DNA lesions. One theory states that charge transfer is involved in protein cross talking through ds-DNA. For these reasons a comparative analysis of ds-oligo containing a mismatched base pair dA:::dG and a damaged dA::dG OXO is proposed. Additionally, the electronic properties of the short ds-oligo in the context of non-equilibrated and equilibrated solvent modes were taken into theoretical consideration. All energetic calculations were performed at the M062x/6-31++G** level of theory, while for geometry optimized ONIOM methodology was used. The lowest adiabatic ionization potential was assigned for DNA containing a dA:dG OXO pair. Moreover, the adiabatic electron affinity was assigned at the same level for the mismatched and lesioned ds-oligo. Surprisingly, in the non-equilibrated mode, a significantly higher vertical electro affinity was found for lesioned DNA. The higher VEA in a non-equilibrated solvent state supported faster recognition in the A:G OXO base pair than A:G by MutY glycosylases under electron transfer mechanism., (Copyright © 2021. Published by Elsevier Ltd.)

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.

DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate ( k pol ), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.

In this study, we incorporated 8-trifluoromethyl-2'-deoxyguanosine ( F G) into a thrombin binding aptamer (TBA). Circular dichroism, nuclear magnetic resonance (NMR), electrophoresis, and prothrombin time (PT) assay were performed to investigate the structure, thermodynamic stability, biological stability, and anticoagulant activity of the F G-modified TBA sequences. We found that the replacement of F G into TBA sequences led to a remarkable improvement in the melting temperature up to 30 °C compared with the native sequence. The trifluoromethyl group allowed us to investigate the TBA G-quadruplex structure by 19 F NMR spectroscopy. Furthermore, PT assays showed that the modified sequences can significantly improve the anticoagulant activity in comparison with the native TBA. Finally, we demonstrated that the trifluoromethyl-modified TBA sequence could function as an anticoagulant reagent in live rats. Our results strongly suggested that F G is a powerful nucleoside derivative to increase the thermodynamic stability and anticoagulant activity of TBA.

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.)

DNA reacts directly with UV light with a wavelength shorter than 300 nm. Although ground surface sunlight includes little of this short-wavelength UV light due to its almost complete absorption by the atmosphere, sunlight is the primary cause of skin cancer. Photosensitization by endogenous substances must therefore be involved in skin cancer development mechanisms. Uric acid is the final metabolic product of purines in humans, and is present at relatively high concentrations in cells and fluids. When a neutral mixed solution of 2'-deoxycytidine, 2'-deoxyguanosine, thymidine, and 2'-deoxyadenosine was irradiated with UV light with a wavelength longer than 300 nm in the presence of uric acid, all the nucleosides were consumed in a uric acid dose-dependent manner. These reactions were inhibited by the addition of radical scavengers, ethanol and sodium azide. Two products from 2'-deoxycytidine were isolated and identified as N 4 -hydroxy-2'-deoxycytidine and N 4 ,5-cyclic amide-2'-deoxycytidine, formed by cycloaddition of an amide group from uric acid. A 15 N-labeled uric acid, uric acid-1,3- 15 N 2 , having two 14 N and two 15 N atoms per molecule, produced N 4 ,5-cyclic amide-2'-deoxycytidine containing both 14 N and 15 N atoms from uric acid-1,3- 15 N 2 . Singlet oxygen, hydroxyl radical, peroxynitrous acid, hypochlorous acid, and hypobromous acid generated neither N 4 -hydroxy-2'-deoxycytidine nor N 4 ,5-cyclic amide-2'-deoxycytidine in the presence of uric acid. These results indicate that uric acid is a photosensitizer for the reaction of nucleosides by UV light with a wavelength longer than 300 nm, and that an unidentified radical derived from uric acid with a delocalized unpaired electron may be generated.

G-quadruplexes are non-canonical four stranded secondary structures possessing great biological importance. Controlling G-quadruplex conformation for further regulating biological processes is both exciting and challenging. In this study, we described a method for regulating G-quadruplex conformation by click chemistry for the first time. 8-ethynyl-2'-deoxyguanosine was synthesized and incorporated into a 12-nt telomere DNA sequence. Such a sequence, at first, formed mixed parallel/anti-parallel G-quadruplexes, while it changed to anti-parallel after reaction with azidobenzene. Meanwhile, the click reaction can give the sequence intense fluorescence.

Product analysis from the iron Fenton oxidation of 2'-deoxyguanosine found reactions in bicarbonate buffer yield 8-oxo-2'-deoxyguanosine and spiroiminodihyantoin consistent with CO3˙-. Reactions in phosphate buffer furnished high yields of sugar oxidation products consistent with HO˙. These observations change the view of DNA oxidation products from the iron-Fenton reaction.

The dA::dG oxo pair appearing in nucleic ds-DNA can lead to a mutation in the genetic information. Depending on the dG oxo source, an AT→GC and GC→AC transversion might be observed. As a result, glycosylases are developed during the evolution, i.e., OGG1 and MutY. While the former effectively removes G oxo from the genome, the second one removes adenine from the dA::dG oxo and dA:dG pair. However, dA::dG oxo is recognized by MutY as ~6-10 times faster than dA:dG. In this article, the structural and electronic properties of simple nucleoside pairs dA:dG, dC:::dG oxo , dC:::dG, dA::dG oxo in the aqueous phase have been taken into theoretical consideration. The influence of solvent relaxation on the above is also discussed. It can be concluded that the dA::dG oxo nucleoside pair shows a lower ionization potential and higher electron affinity than the dA:dG pair in both a vertical and adiabatic mode. Therefore, it could be predicted, under electronic properties, that the electron ejected, for instance by a MutY 4[Fe-S] 2+ cluster, is predisposed to trapping by the ds-DNA part containing the dA::dG oxo pair rather than by dA::dG.

Quenching by nucleobases can significantly affect the fluorescence properties of many fluorophores. The quenching efficiency depends on the electronic properties of the fluorophore and adjacent nucleobases. In this study, we present a hitherto unreported high-efficiency quenching (up to 90%) of various fluorescently labeled pyrrolidinyl peptide nucleic acid (acpcPNA) probes by oligodeoxyguanosine (dGX). The quenching principle relies on the electrostatic interaction between the positively charged lysine-modified acpcPNA probe and the negatively charged oligodeoxyguanosine. The addition of stoichiometric quantities of a DNA target with the sequence complementary to the PNA probe restored the fluorescence to the original level. This was explained by the binding of the DNA to the PNA via a specific base pairing, which resulted in the separation of the oligodeoxyguanosine quencher from the fluorophore. Much less fluorescence restoration was observed in the DNA containing one or more mismatched bases. Applications of the oligodeoxyguanosine-quenched PNA probes for DNA sequence determination, including in multiplex formats, are demonstrated. The performance in terms of sensitivity and mismatch discrimination is comparable to classical PNA-based molecular beacons but without the need for double-labeling, which is expensive and presents solubility issues, or a dedicated quencher probe. This exemplifies a novel use of the unique electrostatic properties of PNA to develop a DNA sensing platform for DNA sequence determination.

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles as well as being related to cancer and other genetic diseases. It is therefore important to investigate Z-DNA structure and related biological events in living cells. However, the development of molecular probes for the observation of Z-DNA structures inside living cells has not yet been realized. Here, we have succeeded in developing site-specific trifluoromethyl oligonucleotide DNA by incorporation of 8-trifluoromethyl-2'-deoxyguanosine (FG). 2D NMR strongly suggested that FG adopted a syn conformation. Trifluoromethyl oligonucleotides dramatically stabilized Z-DNA, even under physiological salt concentrations. Furthermore, the trifluoromethyl DNA can be used to directly observe Z-form DNA structure and interaction of DNA with proteins in vitro, as well as in living human cells by19F NMR spectroscopy for the first time. These results provide valuable information to allow understanding of the structure and function of Z-DNA., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The deuteration of N 2 -ethyl-2'-deoxyguanosine (Et-dG), which is a DNA adduct generated from acetaldehyde, was studied by the addition reaction of acetaldehyde-d 4 to 2'-deoxyguanosine (dG) in deuterium oxide (D 2 O), with the aim to obtain an isotope internal standard for the liquid chromatography/tandem mass spectrometry (LC/MS/MS) quantitation of Et-dG. The replacement of the dG C-8 hydrogen atom by a deuteron atom took place at 50°C in D 2 O and afforded a mixture of Et-dG-d 4 and Et-dG-d 5 . Et-dG-d 4 , which was stable in aqueous solutions, was prepared by incubating the mixture in H 2 O at 60°C for 48 h. The calibration curve was obtained by multiple reaction monitoring (MRM) measurements using a hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometric (HILIC/ESI-MS/MS) system between the Et-dG concentration, ranging from 1.0 × 10 -10 to 4.0 × 10 -9 M in the sample solutions, and the relative peak areas of Et-dG (m/z: 296.1 → 180.1) to the value of Et-dG-d 4 (m/z: 300.2 → 184.2), with an internal standard showing good linearity (R 2 = 0.995, n = 5).

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.)

N 6 -(2-Deoxy-α,β-d-erythropentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy⋅dG) is a major DNA lesion produced from 2'-deoxyguanosine under oxidizing conditions. Fapy⋅dG is produced from a common intermediate that leads to 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo), and in greater quantities in cells. The impact of Fapy⋅dG on DNA structure and function is much less well understood than that of 8-OxodGuo. This is largely due to the significantly greater difficulty in synthesizing oligonucleotides containing Fapy⋅dG than 8-OxodGuo. We describe a synthetic approach for preparing oligonucleotides containing Fapy⋅dG that will facilitate intensive studies of this lesion in DNA. A variety of oligonucleotides as long as 30 nucleotides are synthesized. We anticipate that the chemistry described herein will provide an impetus for a wide range of studies involving Fapy⋅dG., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS 3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O 4 -alkylthymidine ( O 4 -alkyldT), O 2 -alkyldT, O 6 -alkyl-2'-deoxyguanosine ( O 6 -alkyldG), and N 2 -alkyldG through MS 3 analysis. The MS 3 of O 2 - and O 4 -MedT exhibit different fragmentation patterns from each other and from other O 2 - and O 4 -alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O 2 -alkyldT, O 4 -alkyldT, and O 6 -alkyldG adducts carrying larger alkyl groups, whereas O 6 -MedG mainly undergoes elimination of ammonia. The breakdown of N 2 -alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.

Density functional theory (DFT) calculations of reactions between 2'-deoxyguanosine (dR-Gua) and hydroxyl radical (HO • ) with water molecules (H 2 O) n , n = 0, 1, and 2, were carried out. The HO • addition to three carbon sites, C(4), C(5), and C(8), and the subsequent ring cleavage of the three HO adducts were investigated. The addition to C(5) is of the smallest activation energy according to the largest lobe of the dR-Gua highest occupied molecular orbital (HOMO) at C(5). However, its adduct has small stability, and the C(8) adduct has the largest one. The C(8) adduct and the ring-opened amide have similar stability, which would lead to the apparent small yield of the former. Calculations were also performed on HO • additions to the C(4) and C(8) sites of the guanine moiety of four dimer sequence models of B-form DNA with nucleotide moieties (a) 5'-GA-3', (b) 5'-GG-3', (c) 5'-GT-3', and (d) 5'-GC-3'. For instance, the (a) 5'-GA-3' model has a molecular formula C 39 H 50 N 15 Na 2 O 21 P 2 . The HO • attack to C(4) is ruled out owing to the reinforced deformation of the parallel stacking of base pairs. The clear selectivity that the (b) 5'-GG-3' sequence is most reactive was found with the inclusion of the water dimer.

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 C8-aryl 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 Nar I 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.

The fluorescent base guanine analog, 8-vinyl-deoxyguanosine (8vdG), is studied in solution using a combination of optical spectroscopies, notably femtosecond fluorescence upconversion and quantum chemical calculations, based on time-dependent density functional theory (TD-DFT) and including solvent effect by using a mixed discrete-continuum model. In all investigated solvents, the fluorescence is very long lived (3-4 ns), emanating from a stable excited state minimum with pronounced intramolecular charge-transfer character. The main non-radiative decay channel features a sizeable energy barrier and it is affected by the polarity and the H-bonding properties of the solvent. Calculations provide a picture of dynamical solvation effects fully consistent with the experimental results and show that the photophysical properties of 8vdG are modulated by the orientation of the vinyl group with respect to the purine ring, which in turn depends on the solvent. These findings may have importance for the understanding of the fluorescence properties of 8vdG when incorporated in a DNA helix., Competing Interests: The authors declare no conflict of interest.

Cyclic-1,N 2 -propano-2'-deoxyguanosine-d 7 (CPr-dG-d 7 ) was prepared as an isotopic internal standard (IS) for electrospray ionization tandem mass spectrometry (ESI-MS/MS) quantification of CPr-dG in DNA as a candidate cancer risk marker of acetaldehyde intake, mainly from drinking. The deuterated compound was reasonably synthesized from acetaldehyde-d 4 and 2'-deoxyguanosine in deuterium oxide (D 2 O), preventing the deuterium atoms of acetaldehyde-d 4 from being substituted by hydrogen atoms, which occurred seriously in aqueous synthesis media via keto-enol tautomerism. Furthermore, another deuterium atom was added from D 2 O to form CPr-dG-d 7 . After four weeks of storage in H 2 O at 10°C, CPr-dG-d 7 was found to be sufficiently stable for practical use. The calibration curve of CPr-dG by using a hydrophilic interaction chromatography-ESI-MS/MS system with CPr-dG-d 7 as the IS showed sufficient linearity from 1.0 × 10 -10 to 4.0 × 10 -9 M with r 2 = 0.998.

A recent bioinformatic analysis of well-characterized classes of riboswitches uncovered subgroups unable to bind to the regulatory molecule of the parental class. Within the guanine/adenine class, seven groups of RNAs were identified that deviate from the consensus sequence at one or more of three positions directly involved purine nucleobase recognition, one of which was validated as a second class of 2'-deoxyguanosine riboswitch (called 2'-dG-II). To understand how 2'-dG-II riboswitches recognize their cognate ligand and how they differ from a previously identified class of 2'-deoxyguanosine binding riboswitches, we have solved the crystal structure of a 2'-dG-II aptamer domain bound to 2'-deoxyguanosine. This structure reveals a global architecture similar to other members of the purine riboswitch family, but contains key differences within the ligand binding core. Defining the 2'-dG-II riboswitches is a two-nucleotide insertion in the three-way junction that promotes novel base-base interactions. Unlike 2'-dG-I riboswitches, the 2'-dG-II class only requires local changes to the ligand binding pocket of the guanine/adenine class to achieve a change in ligand preference. Notably, members of the 2'-dG-II family have variable ability to discriminate between 2'-deoxyguanosine and riboguanosine, suggesting that a subset of 2'-dG-II riboswitches may bind either molecule to regulate gene expression., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.

N7-Methyl-2'-deoxyguanosine (MdG) is the major damage product in DNA produced by methylating agents, but it often thought to be nontoxic and nonmutagenic. MdG is chemically unstable. An abasic site (AP) is the major product produced from MdG under physiologically relevant conditions. AP formation is frequently considered to be responsible for the cytotoxic effects of MdG, but the reaction is suppressed in nucleosome core particles (NCPs). Recently, it was discovered that histone proteins form reversible DNA-protein cross-links (DPCs) with MdG in reconstituted NCPs, as well as in methylmethanesulfonate (MMS) treated cells. In this study, the formation and reactivity of MdG in MMS treated NCPs was examined at single nucleotide resolution. Sequences consisting of three or more consecutive dGs are more reactive with MMS. The efficiency and selectivity of MdG formation by MMS is largely unaffected within a NCP, although reactivity at several dGs is ∼1.5-2.5-fold higher in NCPs. DPC formation from MdG (DPC MdG ) predominates over AP at all positions within the NCP. With few exceptions, DPC MdG yield is strongly dependent upon the accessibility of the major groove containing MdG to lysine-rich histone N-terminal tails. These data indicate that histone-MdG DPC formation will depend upon DNA sequence and translational position within an NCP.

Quinones may be formed metabolically or abiotically from environmental pollutants and polycyclic aromatic hydrocarbons (PAHs); many are recognized as toxicological intermediates that cause a variety of deleterious cellular effects including mutagenicity. The PAH- o -quinone, 1,2-naphthoquinone (1,2-NQ), may exert its genotoxic effects through interactions with cellular nucleophiles such as DNA, however, the mechanisms of 1,2-NQ adduct formation are still under investigation. With the aim to further understand these mechanisms, the chemical structures of adducts formed from the reaction of 2'-deoxyguanosine (dG) with 1,2-NQ under physiological conditions were investigated by liquid chromatography electrospray ionization tandem mass spectrometry and 1 H NMR analyses. Results showed that 1,2-NQ underwent non-enzymatic oxidation to form a 1,2-NQ-epoxide which in turn formed at least four bulky adducts with dG, and these adducts were more likely to be formed under physiological conditions. A mechanism was proposed whereby hydration of 1,2-NQ to form unstable naphthohydroquinones and 2-hydroxy-1,4-naphthoquinone resulted in formation of hydrogen peroxide that oxidized 1,2-NQ. These results suggest that the genotoxicity of 1,2-NQ may not only be caused through oxidative DNA damage and adduct formation through Michael addition but also through non-enzymatic oxidative transformation of 1,2-NQ itself to form an intermediate PAH-epoxide which covalently binds to DNA.

The N 2 -position of 2'-deoxyguanosine (N 2 -position in dG) is well known for forming carcinogenic minor groove DNA adducts, which originate from environmental pollutants, chemicals, and tobacco smoke. The N 2 -dG DNA adducts have strong implications on biological processes such as DNA replication and repair and may, therefore, result in genomic instability by generating mutations or even cell death. It is crucial to know the role of DNA polymerases when they encounter the N 2 -dG damaged site in DNA. To get detailed insights on the in vitro DNA damage tolerance or bypass mechanism, there is a need to synthetically access N 2 -dG damaged DNAs. This article describes a detailed protocol of the synthesis of N 2 -aryl-dG modified nucleotides using the Buchwald-Hartwig reaction as a main step and incorporation of the modified nucleotides into DNA. In Basic Protocol 1, we focused on the synthesis of five different N 2 -dG modified phosphoramidites with varying bulkiness (benzyl to pyrenyl). Basic Protocol 2 describes the details of synthesizing N 2 -dG modified oligonucleotides employing the standard solid phase synthesis protocol. This strategy provides robust synthetic access to various modifications at the N 2 -position of dG; the modified dGs serve as good substrates to study translesion synthesis and repair pathways. Overall data presented in this article are based on earlier published reports. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Nucleoside configuration (α-d vs. β-d), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2'-deoxyguanosines and anomeric 2'-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2'-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d/α-d and β-d/β-d homo base pairs or α-d/β-d and β-d/α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective T m stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d/β-d or α-d/α-d nucleoside combinations are more stable than α-d/β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

PAGE and UV melting analysis revealed that longer LNA-based splice-switching oligonucleotides (SSOs) formed secondary structures by themselves, reducing their effective concentration. To avoid such secondary structure formation, we introduced 7-deaza-2'-deoxyguanosine or 2'-deoxyinosine into the SSOs. These modified SSOs, with fewer secondary structures, showed higher exon skipping activities.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N 2 -etheno-2'-deoxyguanosine (1,N 2 -εdGuo) and 1,N 6 -etheno-2'-deoxyadenosine (1,N 6 -εdAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32 P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present. We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N 6 -dAdo and 1,N 2 -dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

Reactive oxygen species (ROS) accumulation is known to induce carcinogenesis and accelerate cancer progression. 8‑Hydroxydeoxyguanosine (8‑OHdG) is a specific marker of ROS‑mediated DNA damage. Therefore, we analysed 8‑OHdG levels in cancerous and normal tissue DNA via enzyme‑linked immunosorbent assay (ELISA) using 97 tissue specimens obtained from surgically‑treated patients with stage II/III colorectal cancer (CRC). Additionally, 8‑OHdG levels in these tissues were also assessed via quantitative immunohistochemistry (qIHC). To eliminate individual background variables, the ratio of 8‑OHdG levels between cancerous and normal tissues was calculated using both techniques. A comparative analysis demonstrated that the 8‑OHdG ratio in DNA was significantly correlated with both lymph node metastasis and lymphatic invasion. Multivariate analysis revealed that a high 8‑OHdG ratio in DNA was independently correlated with poor prognosis. These results suggest that the 8‑OHdG ratio in DNA reflects ROS‑induced cancer progression. Conversely, a low 8‑OHdG ratio as estimated via qIHC was an independent factor for poor prognosis. In Kaplan‑Meier analysis, the combination of a high 8‑OHdG ratio in DNA (ELISA) and a low 8‑OHdG ratio in cytoplasm (qIHC) was associated with markedly worse patient prognosis than other combinations. Combined evaluation of the 8‑OHdG ratio using ELISA and qIHC may be pivotal for predicting surgical outcomes for patients with stage II/III CRC.