Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric <i>O</i>²-, <i>N</i>3-, and <i>O</i>⁴-ethylthymidine (<i>O</i>²-, <i>N</i>3-, and <i>O</i>⁴-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf^–) of <i>Escherichia coli</i> DNA polymerase I. Our results showed that hPol η and Kf^– could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove <i>N</i>²-substituted 2′-deoxyguanosine lesions, hPol κ was able to bypass <i>O</i>²-EtdT, though it experienced great difficulty in bypassing <i>N</i>3-EtdT and <i>O</i>⁴-EtdT. yPol ζ was only modestly blocked by <i>O</i>⁴-EtdT, but the polymerase was strongly hindered by <i>O</i>²-EtdT and <i>N</i>3-EtdT. LC–MS/MS analysis of the replication products revealed that DNA synthesis opposite <i>O</i>⁴-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of <i>O</i>²-EtdT and <i>N</i>3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf^–, dTMP. Thus, our results suggested that <i>O</i>²-EtdT and <i>N</i>3-EtdT may also contribute to the AT → TA and AT → GC mutations observed in cells and tissues of animals exposed to ethylating agents

Replicative Bypass of <i>O</i>²‑Alkylthymidine Lesions <i>in Vitro</i>

DNA alkylation represents a major type of DNA damage and is generally unavoidable due to ubiquitous exposure to various exogenous and endogenous sources of alkylating agents. Among the alkylated DNA lesions, <i>O</i>²-alkylthymidines (<i>O</i>²-alkyldT) are known to be persistent and poorly repaired in mammalian systems and have been shown to accumulate in the esophagus, lung, and liver tissue of rats treated with tobacco-specific <i>N</i>-nitrosamines, i.e., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and <i>N</i>′-nitrosonornicotine (NNN). In this study, we assessed the replicative bypass of a comprehensive set of <i>O</i>²-alkyldT lesions, with the alkyl group being a Me, Et, <i>n</i>Pr, <i>i</i>Pr, <i>n</i>Bu, <i>i</i>Bu, or <i>s</i>Bu, in template DNA by conducting primer extension assays with the use of major translesion synthesis DNA polymerases. The results showed that human Pol η and, to a lesser degree, human Pol κ, but not human polymerase ι or yeast polymerase ζ, were capable of bypassing all <i>O</i>²-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that human Pol η exhibited high frequencies of misincorporation of dCMP opposite those <i>O</i>²-alkyldT lesions bearing a longer straight-chain alkyl group. However, the nucleotide misincorporation opposite branched-chain lesions was not selective, with dCMP, dGMP, and dTMP being inserted at similar efficiencies, though the total frequencies of nucleotide misincorporation opposite the branched-chain lesions differed and followed the order of <i>O</i>²-<i>i</i>PrdT > <i>O</i>²-<i>i</i>BudT > <i>O</i>²-<i>s</i>BudT. Together, the results from the present study provided important knowledge about the effects of the length and structure of the alkyl group in the <i>O</i>²-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η

Cytotoxic and Mutagenic Properties of C3′-Epimeric Lesions of 2′-Deoxyribonucleosides in <i>Escherichia coli</i> Cells

Reactive oxygen species (ROS), resulting from endogenous metabolism and/or environmental exposure, can induce damage to the 2-deoxyribose moiety in DNA. Specifically, a hydrogen atom from each of the five carbon atoms in 2-deoxyribose can be abstracted by hydroxyl radical, and improper chemical repair of the ensuing radicals formed at the C1′, C3′, and C4′ positions can lead to the stereochemical inversion at these sites to yield epimeric 2-deoxyribose lesions. Although ROS-induced single-nucleobase lesions have been well studied, the biological consequences of the C3′-epimeric lesions of 2′-deoxynucleosides, i.e., 2′-deoxyxylonucleosides (dxN), have not been comprehensively investigated. Herein, we assessed the impact of dxN lesions on the efficiency and fidelity of DNA replication in <i>Escherichia coli</i> cells by conducting a competitive replication and adduct bypass assay with single-stranded M13 phage containing a site-specifically incorporated dxN. Our results revealed that, of the four dxN lesions, only dxG constituted a strong impediment to DNA replication, and intriguingly, dxT and dxC conferred replication bypass efficiencies higher than those of the unmodified counterparts. In addition, the three SOS-induced DNA polymerases (Pol II, Pol IV, and Pol V) did not play any appreciable role in bypassing these lesions. Among the four dxNs, only dxA directed a moderate frequency of dCMP misincorporation. These results provided important insights into the impact of the C3′-epimeric lesions on DNA replication in <i>E. coli</i> cells

Quantification of Azaserine-Induced Carboxymethylated and Methylated DNA Lesions in Cells by Nanoflow Liquid Chromatography-Nanoelectrospray Ionization Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method

Humans are exposed to <i>N</i>-nitroso compounds through environmental exposure and endogenous metabolism. Some <i>N</i>-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure. In this study, we developed a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization-multistage tandem mass spectrometry (nLC-nESI-MS³) method for the simultaneous quantification of <i>O</i>⁶-carboxymethyl-2′-deoxyguanosine (<i>O</i>⁶-CMdG), <i>O</i>⁶-methyl-2′-deoxyguanosine (<i>O</i>⁶-MedG), and <i>N</i>⁶-carboxymethyl-2′-deoxyadenosine (<i>N</i>⁶-CMdA). We were able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. Our results revealed that the levels of <i>O</i>⁶-CMdG and <i>O</i>⁶-MedG increased when the dose of azaserine was increased from 0 to 450 μM. We, however, did not observe an apparent dose-dependent induction of <i>N</i>⁶-CMdA, suggesting the presence of repair mechanism(s) for the rapid clearance of this lesion in cells. This is the first report about the application of nLC-nESI-MS³ technique for the simultaneous quantification of <i>O</i>⁶-CMdG, <i>O</i>⁶-MedG, and <i>N</i>⁶-CMdA. The method reported here will be useful for future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis

Molecular Design and Property Prediction for a Series of Novel Dicyclic Cyclotrimethylene Trinitramines (RDX) Derivatized as High Energy Density Materials

Quantum chemistry calculations and thermodynamics methods were carried out to screen out novel high energy density materials (HEDMs) from several new derivatives with dicyclic structures of Cyclotrimethylene trinitramine (RDX). Their volumes, densities, heats of formation, detonation properties and impact sensitivities have been calculated with thermodynamics methods under DFT B3LYP 6-31++g (d, p) level and all of these compounds exhibit good performance as HEDMs. Especially, R4 has given outstanding values as a potential HEDM. Its crystal density (2.07 g/cm³), heat of detonation (1.67 kJ/g), detonation velocity (10051m/s), and detonation pressure (48.5 GPa) are even higher than those of CL-20 while its impact sensitivity (<i>h</i>₅₀, 16 cm) remains a relative safety value. The results indicate that the derivative work in common explosives is a good strategy which can design novel HEDMs with high energetic properties and low sensitivity. And furthermore, some mature processes can be used to synthesize them

A Green and Effective Approach of Two-Step 2,2′,4,4′,6,6′-Hexanitrostilbene Preparation and Its Industrial Scale Study

An environmental friendly approach for two-step synthesis of hexanitrostilbene (HNS) has been studied here. In the first step from trinitrotoluene (TNT) to hexanitrobienzyl (HNBB), commercial NaClO was employed as oxidant in mixed solvent of ethyl acetate/ethanol (0.25 mL/1.25 mL per mmol of TNT) instead of benzene/ethanol. In the second step from HNBB to HNS, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)/FeCl₂ was used as an effective catalytic system with O₂ in DMSO solvent. A complex of metal ion and O₂ [M­(<i>n</i>+1)­OO•] was supposed to be the active agent, and TEMPO itself was difficult to initiate dehydrogenation of HNBB but could promote the catalytic cycle of complex M­(<i>n</i>+1)­OO•. Finally, we increased the scale from 30 to 1000 g to investigate the feasibility of production. The total yield of two steps would be unprecedentedly as high as 70%

Effects of Tet-Induced Oxidation Products of 5‑Methylcytosine on DNA Replication in Mammalian Cells

Recently 5-hydroxymethyl-2′-deoxycytidine (5hmdC), 5-formyl-2′-deoxycytidine (5fdC), and 5-carboxyl-2′-deoxycytidine (5cadC) were discovered in mammalian DNA as oxidation products of 5-methyl-2′-deoxycytidine (5mdC) induced by the ten-eleven translocation family of enzymes. These oxidized derivatives of 5mdC may not only act as intermediates of active cytosine demethylation in mammals but also serve as epigenetic marks on their own. It remains unclear how 5hmdC, 5fdC, and 5cadC affect DNA replication in mammalian cells. Here, we examined the effects of the three modified nucleosides on the efficiency and accuracy of DNA replication in HEK293T human kidney epithelial cells. Our results demonstrated that a single, site-specifically incorporated 5fdC or 5cadC conferred modest drops, by approximately 30%, in replication bypass efficiency without inducing detectable mutations in human cells, whereas replicative bypass of 5hmdC is both accurate and efficient. The lack of pronounced perturbation of these oxidized 5mdC derivatives on DNA replication is consistent with their roles in epigenetic regulation of gene expression

<i>In Vitro</i> Lesion Bypass Studies of <i>O</i>⁴‑Alkylthymidines with Human DNA Polymerase η

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, <i>O</i>⁴-alkylthymidine (<i>O</i>⁴-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various <i>O</i>⁴-alkyldT lesions, with the alkyl group being Me, Et, <i>n</i>Pr, <i>i</i>Pr, <i>n</i>Bu, <i>i</i>Bu, (<i>R</i>)-<i>s</i>Bu, or (<i>S</i>)-<i>s</i>Bu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all <i>O</i>⁴-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite <i>O</i>⁴-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite <i>O</i>⁴-<i>i</i>PrdT, <i>O</i>⁴-<i>i</i>BudT, and <i>O</i>⁴-(<i>R</i>)-<i>s</i>BudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in <i>O</i>⁴-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η

Table_1_Transcriptomic analysis reveals the potential crosstalk genes and immune relationship between IgA nephropathy and periodontitis.docx

BackgroundIt is well known that periodontitis has an important impact on systemic diseases. The aim of this study was to investigate potential crosstalk genes, pathways and immune cells between periodontitis and IgA nephropathy (IgAN).MethodsWe downloaded periodontitis and IgAN data from the Gene Expression Omnibus (GEO) database. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were used to identify shared genes. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the shared genes. Hub genes were further screened using least absolute shrinkage and selection operator (LASSO) regression, and a receiver operating characteristic (ROC) curve was drawn according to the screening results. Finally, single-sample GSEA (ssGSEA) was used to analyze the infiltration level of 28 immune cells in the expression profile and its relationship with shared hub genes.ResultsBy taking the intersection of WGCNA important module genes and DEGs, we found that the SPAG4, CCDC69, KRT10, CXCL12, HPGD, CLDN20 and CCL187 genes were the most important cross-talk genes between periodontitis and IgAN. GO analysis showed that the shard genes were most significantly enriched in kinase regulator activity. The LASSO analysis results showed that two overlapping genes (CCDC69 and CXCL12) were the optimal shared diagnostic biomarkers for periodontitis and IgAN. The immune infiltration results revealed that T cells and B cells play an important role in the pathogenesis of periodontitis and IgAN.ConclusionThis study is the first to use bioinformatics tools to explore the close genetic relationship between periodontitis and IgAN. The SPAG4, CCDC69, KRT10, CXCL12, HPGD, CLDN20 and CCL187 genes were the most important cross-talk genes between periodontitis and IgAN. T-cell and B-cell-driven immune responses may play an important role in the association between periodontitis and IgAN.</p

<i>In-Vitro</i> Replication Studies on <i>O</i>²‑Methylthymidine and <i>O</i>⁴‑Methylthymidine

<i>O</i>²- and <i>O</i>⁴-methylthymidine (<i>O</i>²-MdT and <i>O</i>⁴-MdT) can be induced in tissues of laboratory animals exposed with <i>N</i>-methyl-<i>N</i>-nitrosourea, a known carcinogen. These two <i>O</i>-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, <i>in vitro</i> replication studies with duplex DNA substrates containing site-specifically incorporated <i>O</i>²-MdT and <i>O</i>⁴-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of <i>Escherichia coli</i> DNA polymerase I (Kf^–), human DNA polymerase κ (pol κ), and <i>Saccharomyces cerevisiae</i> DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf^– and pol η preferentially incorporated the correct nucleotide (dAMP) opposite <i>O</i>²-MdT, while <i>O</i>⁴-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite <i>O</i>²-MdT and <i>O</i>⁴-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ favorably incorporated the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication <i>in vitro</i>. In addition, the results from our study revealed that, if left unrepaired, <i>O</i>-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents