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The respiratory-chain poison antimycin A promotes the formation of DNA single-strand breaks and reduces toxicity in U937 cells exposed to t-butylhydroperoxide.

ABSTRACT: Antimycin A at levels that abolish oxygen consumption had a slight, although statistically significant, inhibitory effect on the toxicity elicited by t-butylhydroperoxide in U937 cells. The protective effect was observed after 6 h of post-treatment incubation, but was no longer apparent after 24 h. Unexpectedly, these events were associated with a marked accumulation of DNA single-strand breaks produced by low levels of t-butylhydroperoxide. Both an oxygen- and a carbon-centred radical were found to arise during treatment with t-butylhydroperoxide, and their formation was significantly lowered by antimycin A. Thus inhibition of electron transport at the level of complex III appears (a) to decrease the formation of toxic species which mediate, at least partially, the lethal effects elicited by t-butylhydroperoxide, and (b) to enhance the formation of DNA-damaging species generated at low concentrations of t-butylhydroperoxide. Rotenone and cyanide, which respectively inhibit complexes I and IV, did not affect DNA damage elicited by t-butylhydroperoxide. These results suggest that DNA single-strand breaks do not mediate the toxicity of t-butylhydroperoxide, and that specific mitochondrial functions might modulate the formation of the toxic and of DNA-damaging species generated by organic hydroperoxides.

SUBMITTER: Guidarelli A

PROVIDER: S-EPMC1217497 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template.

Project description:Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI 'cleavase', but no evidence of toxicity with the I-AniI 'nickase.' Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs.

| S-EPMC3035452 | biostudies-literature

Induction of DNA double-strand breaks and cellular senescence by human respiratory syncytial virus.

Project description:Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract infections during infancy and childhood and is associated with significant morbidity and mortality. HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and γH2AFX in cultured cells secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci contained γH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester (GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs of DNA damage and aging such as γH2AFX and CDKN2A expression in the respiratory epithelia of infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA damage-mediated cellular senescence program probably mediated by oxidative stress. The results also suggest that this program might contribute to the physiopathology of the infection, tissue remodeling and aging, and might be associated to long-term consequences of HRSV infections.

| S-EPMC4871660 | biostudies-literature

Intracellular ascorbic acid enhances the DNA single-strand breakage and toxicity induced by peroxynitrite in U937 cells.

Project description:A well-established protocol to increase the intracellular content of ascorbic acid was used to investigate the effects of the vitamin on DNA single-strand breakage and toxicity mediated by authentic peroxynitrite (ONOO(-)) in U937 cells. This protocol involved exposure for 60 min to 100 microM dehydroascorbic acid, which was taken up by the cells and converted into ascorbic acid via a GSH-independent mechanism. At the time of exposure to ONOO(-), which was performed in fresh saline immediately after loading with dehydroascorbic acid, the vitamin present in the cells was all in its reduced form. It was found that, in cells that are otherwise ascorbate-deficient, an increase in their ascorbic acid content does not prevent, but rather enhances, the DNA-damaging and lethal responses mediated by exogenous ONOO(-). These results therefore suggest that acute supplementation of ascorbic acid can be detrimental for individuals with pathologies associated with a decrease in ascorbic acid and in which ONOO(-) is known to promote deleterious effects.

| S-EPMC1221863 | biostudies-other

Synergistic cytotoxicity and DNA strand breaks in cells and plasmid DNA exposed to uranyl acetate and ultraviolet radiation.

Project description:Depleted uranium (DU) has a chemical toxicity that is independent of its radioactivity. The purpose of this study was to explore the photoactivation of uranyl ion by ultraviolet (UV) radiation as a chemical mechanism of uranium genotoxicity. The ability of UVB (302?nm) and UVA (368?nm) radiation to photoactivate uranyl ion to produce single strand breaks was measured in pBR322 plasmid DNA, and the presence of adducts and apurinic/apyrimidinic sites that could be converted to single strand breaks by heat and piperidine was analyzed. Results showed that DNA lesions in plasmid DNA exposed to UVB- or UVA-activated DU were only slightly heat reactive, but were piperidine sensitive. The cytotoxicity of UVB-activated uranyl ion was measured in repair-proficient and repair-deficient Chinese hamster ovary cells and human keratinocyte HaCaT cells. The cytotoxicity of co-exposures of uranyl ion and UVB radiation was dependent on the order of exposure and was greater than co-exposures of arsenite and UVB radiation. Uranyl ion and UVB radiation were synergistically cytotoxic in cells, and cells exposed to photoactivated DU required different DNA repair pathways than cells exposed to non-photoactivated DU. This study contributes to our understanding of the DNA lesions formed by DU, as well as their repair. Results suggest that excitation of uranyl ion by UV radiation can provide a pathway for uranyl ion to be chemically genotoxic in populations with dermal exposures to uranium and UV radiation, which would make skin an overlooked target organ for uranium exposures.

| S-EPMC4428325 | biostudies-literature

Genome-wide mapping of DNA strand breaks.

Project description:Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.

| S-EPMC3045442 | biostudies-literature

Physiological Roles of DNA Double-Strand Breaks.

Project description:Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Among the most cytotoxic lesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix. Cells boast a considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent an important category of anticancer therapeutics. And yet, bizarrely, cells deploy this very machinery for the intentional and calculated disruption of genomic integrity, harnessing potentially destructive DSBs in delicate genetic transactions. Under tight spatiotemporal regulation, DSBs serve as a tool for genetic modification, widely used across cellular biology to generate diverse functionalities, ranging from the fundamental upkeep of DNA replication, transcription, and the chromatin landscape to the diversification of immunity and the germline. Growing evidence points to a role of aberrant DSB physiology in human disease and an understanding of these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs. Here, we review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider how the cell is able to harness DNA breaks as a critical biochemical tool.

| S-EPMC5664317 | biostudies-other

Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern.

Project description:Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28 A resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alphaA helix.

| S-EPMC1482829 | biostudies-literature

In vivo generation of Double strand breaks in hexavalent chromium exposed Drosophila melanogaster larvae

Project description:In vivo transcription profiling in Drosophila melanogaster larvae exposed to hexavalent chromium

2023-05-03 | E-MEXP-3245 | biostudies-arrayexpress

Reading chromatin signatures after DNA double-strand breaks.

Project description:DNA double-strand breaks (DSBs) are DNA lesions that must be accurately repaired in order to preserve genomic integrity and cellular viability. The response to DSBs reshapes the local chromatin environment and is largely orchestrated by the deposition, removal and detection of a complex set of chromatin-associated post-translational modifications. In particular, the nucleosome acts as a central signalling hub and landing platform in this process by organizing the recruitment of repair and signalling factors, while at the same time coordinating repair with other DNA-based cellular processes. While current research has provided a descriptive overview of which histone marks affect DSB repair, we are only beginning to understand how these marks are interpreted to foster an efficient DSB response. Here we review how the modified chromatin surrounding DSBs is read, with a focus on the insights gleaned from structural and biochemical studies.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

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Repair of double-strand breaks by end joining.

Project description:Nonhomologous end joining (NHEJ) refers to a set of genome maintenance pathways in which two DNA double-strand break (DSB) ends are (re)joined by apposition, processing, and ligation without the use of extended homology to guide repair. Canonical NHEJ (c-NHEJ) is a well-defined pathway with clear roles in protecting the integrity of chromosomes when DSBs arise. Recent advances have revealed much about the identity, structure, and function of c-NHEJ proteins, but many questions exist regarding their concerted action in the context of chromatin. Alternative NHEJ (alt-NHEJ) refers to more recently described mechanism(s) that repair DSBs in less-efficient backup reactions. There is great interest in defining alt-NHEJ more precisely, including its regulation relative to c-NHEJ, in light of evidence that alt-NHEJ can execute chromosome rearrangements. Progress toward these goals is reviewed.

| S-EPMC3632057 | biostudies-literature

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