8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

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.

T7 RNA polymerase catalyzed transcription of the epimerizable DNA lesion, Fapy•dG and 8-oxo-2'-deoxyguanosine.

Fapy•dG (N6-(2-deoxy-α,ß-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) are major products of 2'-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer-dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to 2'-deoxyguanosine. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G, or U. Fitting of reaction data indicates that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast- or slow-reacting base pairs vary less than twofold as a function of the nucleotide opposite the lesion. Promoter-dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side by side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

Conformation-dependent lesion bypass of bulky arylamine-dG adducts generated from 2-nitrofluorene in epigenetic sequence contexts.

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.

7-Deaza-2'-deoxyisoguanosine, a Noncanonical Nucleoside for Nucleic Acid Code Expansion and New DNA Constructs: Nucleobase Functionalization of Inverse Watson-Crick and Purine-Purine Base Pairs.

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.

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

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 (DPCMdG), 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 DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG 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, DPCMdG 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.

The Influence of Clustered DNA Damage Containing Iz/Oz and OXOdG on the Charge Transfer through the Double Helix: A Theoretical Study.

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 (OXOdG) 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[A1Iz2A3OXOG4A5]*d[T5C4T3C2T1], and oligo-Oz, d[A1Oz2A3OXOG4A5]*d[T5C4T3C2T1], 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, OXOdG 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 Oz2 to complementary C4 was noted during vertical to adiabatic anion relaxation, while for oligo-Iz, it was settled exclusively on the Iz2 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 sp2 or sp3. The above can confuse the single DNA damage recognition and removal processes, cause an increase in mutagenesis, and harm the effectiveness of anticancer therapy.

The Influence of 2'-Deoxyguanosine Lesions on the Electronic Properties of OXOG:::C Base Pairs in Ds-DNA: A Comparative Analysis of Theoretical Studies.

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 (OXOdG), with an oxidation potential that is 0.56 V lower than that of canonical dG. All this has led to OXOdG, 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 OXOGC 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 OXOG4C2 were largely unaffected by a second lesion. (B) The positive charge and spin were found predominantly on the OXOG4C2 moiety. (C) The electron-hole transfers A3T3→G4C2 and G4C2←A5T1 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 OXOGC 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.

Recognition of 8-Oxo-2'-deoxyguanosine in DNA Using the Triphosphate of 2'-Deoxycytidine Connecting the 1,3-Diazaphenoxazine Unit, dCdapTP.

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.

Base-Displaced Intercalated Structure of the 3-(2-Deoxy-ß-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M1dG) Lesion in DNA.

The genotoxic 3-(2-deoxy-ß-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) 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, M1dG may oxidize, in vivo, to 3-(2-deoxy-ß-D-erythropentofuranosyl)-pyrimido[1,2-f]purine-6,10(3H,5H)-dione (6-oxo-M1dG). 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-M1dG damage in DNA, we conducted NMR studies of 6-oxo-M1dG incorporated site-specifically into 5' -d(C1A2T3X4A5T6G7A8C9G10C11T12)-3':5'-d(A13G14C15G16T17C18A19T20C21A22T23G24)-3' (X = 6-oxo-M1dG). NMR spectra afforded detailed resonance assignments. Chemical shift analyses revealed that nucleobase C21, complementary to 6-oxo-M1dG, was deshielded compared with the unmodified duplex. Sequential NOEs between 6-oxo-M1dG and A5 were disrupted, as well as NOEs between T20 and C21 in the complementary strand. The structure of the 6-oxo-M1dG modified DNA duplex was refined by using molecular dynamics (rMD) calculations restrained by NOE data. It revealed that 6-oxo-M1dG intercalated into the duplex and remained in the anti-conformation about the glycosyl bond. The complementary cytosine C21 extruded into the major groove, accommodating the intercalated 6-oxo-M1dG. The 6-oxo-M1dG H7 and H8 protons faced toward the major groove, while the 6-oxo-M1dG imidazole proton H2 faced into the major groove. Structural perturbations to dsDNA were limited to the 6-oxo-M1dG damaged base pair and the flanking T3:A22 and A5:T20 base pairs. Both neighboring base pairs remained within the Watson-Crick hydrogen bonding contact. The 6-oxo-M1dG did not stack well with the 5'-neighboring base pair T3:A22 but showed improved stacking with the 3'-neighboring base pair A5:T20. Overall, the base-displaced intercalated structure was consistent with thermal destabilization of the 6-oxo-M1dG damaged DNA duplex; thermal melting temperature data showed a 15 °C decrease in Tm compared to the unmodified duplex. The structural consequences of 6-oxo-M1dG formation in DNA are evaluated in the context of the chemical biology of this lesion.

The 2Ih and OXOG Proximity Consequences on Charge Transfer through ds-DNA: Theoretical Studies of Clustered DNA Damage.

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 OXOG 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 OXOG 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.

DNA oxidative damage in oral cancer: 8-hydroxy-2´-deoxyguanosine immunoexpression assessment.

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.

Local Alteration of Ionic Strength in a Nucleosome Core Particle and Its Effect on N7-Methyl-2'-deoxyguanosine Depurination.

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, N7-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.

Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η.

Etheno (ε)-adducts, e.g., 1,N2-ε-guanine (1,N2-ε-G) and 1,N6-ε-adenine (1,N6-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,N2-ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N2-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,N2-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,N2-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,N2-ε-G and T, compared with the single H-bond formed between incoming dCMPnPP and 1,N2-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.

Multi-DNA Adduct and Abasic Site Quantitation In Vivo by Nano-Liquid Chromatography/High-Resolution Orbitrap Tandem Mass Spectrometry: Methodology for Biomonitoring Colorectal DNA Damage.

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/HRAMS2) 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 O6-carboxymethyl-2'-deoxyguanosine, O6-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 108 bases using 3 µg of DNA. Adduct formation in animals ranged from ∼1 in 108 to ∼1 in 105 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-ß-d-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) and 6-oxo-M1dG adduct levels were not increased by carcinogen dosing but increased in FFPE tissues. Human biomonitoring studies can implement LC/HRAMS2 assays for DNA adducts and AP sites outlined in this work to advance our understanding of CRC etiology.

Implications of N7-hydrogen and C8-keto on the base pairing, mutagenic potential and repair of 8-oxo-2'-deoxy-adenosine: Investigation by nucleotide analogues.

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 ß 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.

Oligonucleotides DNA containing 8-trifluoromethyl-2'-deoxyguanosine for observing Z-DNA structure.

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.

Mechanism of Deoxyguanosine Diphosphate Insertion by Human DNA Polymerase ß.

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) ß, we examine how Pol ß 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 ß to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (kpol), 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.

DNA Polymerase η Promotes the Transcriptional Bypass of N2-Alkyl-2'-deoxyguanosine Adducts in Human Cells.

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 N2-alkyl-2'-deoxyguanosine (N2-alkyl-dG, alkyl = methyl, ethyl, n-propyl, or n-butyl) lesions in HEK293T cells. We found that N2-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 N2-alkyl-dG lesions, which is exacerbated by codepletion of Rev1 in Pol η-deficient background. We also observed that the repair of N2-nBu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. Hence, our results provided insights into transcriptional perturbations induced by N2-alkyl-dG lesions and expanded the biological functions of TLS DNA polymerases.

Reactivity and DNA Damage by Independently Generated 2'-Deoxycytidin-N4-yl Radical.

Oxidative stress produces a variety of radicals in DNA, including pyrimidine nucleobase radicals. The nitrogen-centered DNA radical 2'-deoxycytidin-N4-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 O6-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.

Direct Alkylation of Deoxyguanosine by Azaserine Leads to O6-Carboxymethyldeoxyguanosine.

The O6-alkylguanosine adduct O6-carboxymethyldeoxyguanosine (O6-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 O6-CMdG as well as the corresponding methyl adduct O6-methyldeoxyguanosine (O6-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 O6-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 O6-CMdG and O6-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 O6-serine-CMdG (O6-Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O6-Ser-CMdG forms as an intermediate that spontaneously decomposes to form O6-CMdG. Finally, we quantified levels of O6-CMdG and O6-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.