Single nucleotide resolution mapping identifies abasic sites as major oxidation product of oxo8G and as RNA damage marker
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PROVIDER: PRJEB92107 | ENA |
REPOSITORIES: ENA
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Project description:RNA oxidation HOCl
| PRJEB73991 | ENA
Project description:RNA oxidation HOCl Complement
| PRJEB84071 | ENA
Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale.
2012-10-01 | GSE39641 | GEO
Project description:To investigate selectivity of mRNA oxidation, total RNA and oxidized RNA isolated from Neuro 2a cells before and after H2O2 treatment were employed for microarray analysis. It was found that selective oxidation of mRNA already occurs under normal culture conditions but was increased by H2O2, especially in a subset of mRNAs related to certain functions. Moreover, mRNA oxidation level is also related to its abundance or stability (half-life time). This shows for the first time that mRNA oxidation is associated with RNA homeostasis including function, stability and abundance depending on cellular redox status in a genome-wide scale. Neuro 2a cells received hydrogen peroxide treatment or no treatment as a control. Samples were applied for RNA extraction and ARP labeling, which could bind with apurinic/apyrimidinic sites, and then a pull-down process to isolate oxidized RNA. Total RNA and oxidized RNA were used for subsequent transcriptomic profiling. 4 types of samples were analyzed: Basal-total: untreated N2a cells labeled with ARP, but not processed for the pull-down assay. Ox-total: hydrogen peroxide-treated N2a cells labeled with ARP, but not processed for the pull-down assay. Basal-ARP: untreated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis. Ox-ARP: hydrogen peroxide-treated N2a cells labeled with ARP, and processed for the pull-down assay. ARP-derivatized RNA, which is also oxidized RNA, was concentrated and used for the microarray analysis.
2012-10-01 | E-GEOD-39641 | biostudies-arrayexpress
Project description:The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.
| S-EPMC8851688 | biostudies-literature
Project description:RNA is more vulnerable to oxidation than other cellular components, which is linked to a range of diseases and pathological conditions, such as AKI. To identify the contribution of RNA oxidation to AKI, the mechanisms of how cells handle oxidized RNA should be elucidated. By RNA sequencing analysis in ischemia/reperfusion-induced AKI, we observed the significant changes of genes important in degradingting oxidative RNA.
2023-03-03 | GSE226275 | GEO
Project description:Base excision repair (BER) plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ (Pol λ) is believed to play a backup role to DNA polymerase β (Pol β) in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA-damaging agents, including several antitumor antibiotics, the C4'-oxidized abasic site following Ape1 incision (pC4-AP), and 5'-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single-turnover kinetic experiments show that Pol λ excises DOB almost 13 times more slowly than a 5'-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β for the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β, and the difference can be attributed to a higher K(I) (33 ± 7 nM). Inactivation of Pol λ's lyase activity by DOB also prevents the enzyme from conducting polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC-digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support for the idea that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. When acting on pC4-AP, Pol λ achieves approximately four turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity, and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA-damaging agents that produce them.
| S-EPMC3566640 | biostudies-literature
Project description:By relating the numbers of oxidized guanines in different sequence contexts to the abundance of these contexts in the human genome, we observed that the probability of guanine to be or remain oxidized is low if this guanine is preceded by cytosine (CpG sites) compared to other nucleotides. As CpG sites are substrates of epigenetic cytosine methylation, we investigated the influence of cytosine methylation status on guanine oxidation level in CpG sites. For this, we exposed HAP1 and U2OS cells for three days to GSK-3484862, a novel DNA methyltransferase 1 (DNMT1) inhibitor causing the degradation of this enzyme within 24 hours and having low cellular toxicity. Using Infinium MethylationEPIC v2.0 array, we observed a drastic hypomethylation after the exposure in both cell lines such that the peak of highly methylated CpG sites (beta values 0.8–1) found in vehicle (DMSO) exposed cells is absent in GSK-3484862-exposed cells. In contrast, the oxidation level of guanines in the CpG sites profiled by the array did not change in response to the inhibitor and was virtually constant across CpG sites with different methylation levels. In addition, on the level of all CpG sites in the genome, guanine-oxidation did not differ between DMSO and GSK-3484862 exposures. Thus, in CpG sites, guanine oxidation level likely does not depend on cytosine methylation status. As a control for the click-code-seq protocol, we successfully called 8-oxoG incorporated at specific sites in a plasmid (~25 fmol) that was mixed with ~28-times bigger amount of the unmodified vector. As a control for click-code-seq detection of abasic sites, we inserted uracils in a plasmid, converted them to abasic sites by uracil-DNA glycosylase (UDG) and successfully called them in the expected locations.
2025-08-22 | GSE295218 | GEO
Project description:Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance but research into the mechanisms has been stymied by a lack of a genetically tractable pure culture which unequivocally does not use molecular oxygen to activate benzene. Geobacter metallireducens grew in a medium in which benzene was the sole electron donor and Fe(III) was the sole electron acceptor with a stoichiometry of benzene loss and Fe(III) reduction consistent with benzene oxidation to carbon dioxide coupled with Fe(III) reduction. Phenol labeled with 18O was produced when the medium was labeled with H218O, as expected for a true anaerobic conversion of benzene to phenol. Gene expression patterns indicated that benzene was metabolized through a phenol intermediate rather than benzoate or toluene. Deletion of ppcB, which encodes a subunit of the phenylphosphate carboxylase, an enzyme required for phenol metabolism, inhibited metabolism of benzene. Deleting genes specific for benzoate or toluene metabolism did not. Comparison of gene expression patterns in cells grown on benzene versus cells grown on phenol revealed genes specifically expressed in benzene-grown cells. Deletion of one of these, Gmet_3376, inhibited anaerobic benzene oxidation, but not the metabolism of phenol, benzoate, or toluene. The availability of a genetically tractable pure culture that can anaerobically convert benzene to phenol with oxygen derived from water should significantly accelerate elucidation of the mechanisms by which benzene can be activated in the absence of molecular oxygen. Total RNA from three separate cultures of G. metallireducens grown with 250 µM benzene three separate cultures of G. metallireducens grown with 500 µM phenol three separate cultures of G. metallireducens grown with 1 mM benzoate three separate cultures of G. metallireducens grown with 500 µM toluene three separate cultures of G. metallireducens grown with 10 mM acetate were used to study [1] Anaerobic oxidation of benzene by G. metallireducens (Benzene vs. acetate, Benzene vs. benzoate, Benzene vs. phenol, Benzene vs. toluene) [2] Anaerobic oxidation of benzoate by G. metallireducens (Benzoate vs. acetate) [3] Anaerobic oxidation of phenol by G. metallireducens (Phenol vs. acetate) [4] Anaerobic oxidation of toluene by G. metallireducens (Toluene vs. acetate) Each chip measures the expression level of 3,627 genes from G. metallireducens DSM 7210 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
2013-04-30 | E-GEOD-33794 | biostudies-arrayexpress