90 research outputs found
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><sup>2</sup>-, <i>N</i>3-, and <i>O</i><sup>4</sup>-ethylthymidine
(<i>O</i><sup>2</sup>-, <i>N</i>3-, and <i>O</i><sup>4</sup>-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<sup>–</sup>) of <i>Escherichia coli</i> DNA polymerase I. Our results showed that
hPol η and Kf<sup>–</sup> 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><sup>2</sup>-substituted
2′-deoxyguanosine lesions, hPol κ was able to bypass <i>O</i><sup>2</sup>-EtdT, though it experienced great difficulty
in bypassing <i>N</i>3-EtdT and <i>O</i><sup>4</sup>-EtdT. yPol ζ was only modestly blocked by <i>O</i><sup>4</sup>-EtdT, but the polymerase was strongly hindered by <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT. LC–MS/MS
analysis of the replication products revealed that DNA synthesis opposite <i>O</i><sup>4</sup>-EtdT was highly error-prone, with dGMP being
preferentially inserted, while the presence of <i>O</i><sup>2</sup>-EtdT and <i>N</i>3-EtdT in template DNA directed
substantial frequencies of misincorporation of dGMP and, for hPol
ι and Kf<sup>–</sup>, dTMP. Thus, our results suggested
that <i>O</i><sup>2</sup>-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><sup>2</sup>‑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><sup>2</sup>-alkylthymidines (<i>O</i><sup>2</sup>-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><sup>2</sup>-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><sup>2</sup>-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><sup>2</sup>-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><sup>2</sup>-<i>i</i>PrdT > <i>O</i><sup>2</sup>-<i>i</i>BudT > <i>O</i><sup>2</sup>-<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><sup>2</sup>-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-MS<sup>3</sup>) method for the simultaneous quantification of <i>O</i><sup>6</sup>-carboxymethyl-2′-deoxyguanosine (<i>O</i><sup>6</sup>-CMdG), <i>O</i><sup>6</sup>-methyl-2′-deoxyguanosine
(<i>O</i><sup>6</sup>-MedG), and <i>N</i><sup>6</sup>-carboxymethyl-2′-deoxyadenosine (<i>N</i><sup>6</sup>-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><sup>6</sup>-CMdG and <i>O</i><sup>6</sup>-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><sup>6</sup>-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-MS<sup>3</sup> technique for the simultaneous
quantification of <i>O</i><sup>6</sup>-CMdG, <i>O</i><sup>6</sup>-MedG, and <i>N</i><sup>6</sup>-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/cm<sup>3</sup>), 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><sub>50</sub>, 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)/FeCl<sub>2</sub> was used as an effective catalytic system with O<sub>2</sub> in DMSO solvent. A complex of metal ion and O<sub>2</sub> [MÂ(<i>n</i>+1)ÂOO•] 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 MÂ(<i>n</i>+1)ÂOO•.
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><sup>4</sup>‑Alkylthymidines with Human DNA Polymerase η
Environmental exposure and endogenous
metabolism can give rise
to DNA alkylation. Among alkylated nucleosides, <i>O</i><sup>4</sup>-alkylthymidine (<i>O</i><sup>4</sup>-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><sup>4</sup>-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><sup>4</sup>-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><sup>4</sup>-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><sup>4</sup>-<i>i</i>PrdT, <i>O</i><sup>4</sup>-<i>i</i>BudT, and <i>O</i><sup>4</sup>-(<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><sup>4</sup>-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><sup>2</sup>‑Methylthymidine and <i>O</i><sup>4</sup>‑Methylthymidine
<i>O</i><sup>2</sup>- and <i>O</i><sup>4</sup>-methylthymidine (<i>O</i><sup>2</sup>-MdT
and <i>O</i><sup>4</sup>-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><sup>2</sup>-MdT
and <i>O</i><sup>4</sup>-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<sup>–</sup>), 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<sup>–</sup> and pol η preferentially incorporated the correct
nucleotide (dAMP) opposite <i>O</i><sup>2</sup>-MdT, while <i>O</i><sup>4</sup>-MdT primarily directed dGMP misincorporation.
While steady-state kinetic experiments showed that pol κ-mediated
nucleotide insertion opposite <i>O</i><sup>2</sup>-MdT and <i>O</i><sup>4</sup>-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
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