Haut PDF NLRP3 controls ATM activation in response to DNA damage

NLRP3 controls ATM activation in response to DNA damage

NLRP3 controls ATM activation in response to DNA damage

HAL Id: hal-03093041 https://hal.archives-ouvertes.fr/hal-03093041 Preprint submitted on 5 Jan 2021 HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

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Role of ATM in the telomere response to the G-quadruplex ligand 360A.

Role of ATM in the telomere response to the G-quadruplex ligand 360A.

We showed that 360A triggered DNA damage signaling outside telomeres that could be indicative of the formation of G-quadruplex structures at interstitial sites, as pro- posed previously (25,51). This is sustained by the need of functional ATM for this signaling to occur indicating the particularity of these DNA damages. The question of the importance of these interstitial DNA damages for the cellular effects induced by 360A remained asked. However, the telomere targeting by the G-quadruplex ligand is probably the main cause of cell death, since: (i) we did not detect by the methods used a significant level of genetic instability that did not involve telomeres in ATM-proficient, as well as in ATM-deficient cell lines, and (ii) the strong level of telomere instability and the frequent detection of anaphase bridges [data not shown; (24)] observed in treated cells are well consistent with the induction of mitotic catastrophe after few rounds of replication. This is further sustained by the rarity of duplicated telomere doublets on sister chromatids, confirming that the cells did not enter a second cycle of division after induction of telomere recombination regardless their ATM status. However, participation of interstitial DNA damages in telomere recombination could not be excluded. This is suggested particularly in case of TDM and telomere doublets, which are likely due to telomere recombination in cis with interstitial sequences as proposed above.
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Replication Stress, DNA Damage, Inflammatory Cytokines and Innate Immune Response

Replication Stress, DNA Damage, Inflammatory Cytokines and Innate Immune Response

a—SLX4 and MUS81 SLX4 is a platform protein that recruits nucleases that resolve branched DNA intermediates resulting from HR and/or arrested replication fork processing. MUS81 is an endonuclease is an endonuclease that interacts with SLX4 and plays a role in resolving HR intermediates, suppressing chromosomal instability [170]. MUS81 fosters the accumulation of fragmented self-DNA (including DNA lesions, R-loops, repetitive sequences and common fragile sites) resulting from replication stress, leading to STING-dependent expression of type I IFNs and chemokines [171]. SLX4 is mutated in FA Group P. Fibroblasts from FA-P harbour cytoplasmic DNA accumulation, including sequences deriving from active Long INterspersed Element-1 (LINE-1), triggering the cGAS-STING pathway that elicits IFN expression. Similar results were obtained with FA-D2, an upstream activator of SLX4, which caused the accumulation of DNA fragments in the cytoplasm that are recognized by PRRs, inducing an inflammatory response [138] leading to senescence and inhibiting stem cell function [58]. In vitro, stimulated FA bone marrow showed elevated levels of tumour necrosis factor-α (TNF-α) and IFN-γ [172]. Sumpter et al. suggest that FA genes function in the selective autophagy of genetically distinct viruses, in mitochondrial quality control and in preventing inflammasome activation due to mitochondrial reactive oxygen species (ROS) [173].
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DNA damage response upon environmental contaminants: an exhausting work for genomic integrity

DNA damage response upon environmental contaminants: an exhausting work for genomic integrity

AC CE PT ED (including reduction in glucose access). SIRT1 was recently described as a switch of metabolic and DNA damage checkpoints. Precisely, SIRT1 regulates the acetylation/deactelyation of DNA topoisomerase II binding protein 1 (TopBP1) [5]. On the one hand, glucose deprivation induces SIRT1 activation, transducing ToBP1 deacetylation and thereby inhibiting DNA replication. In contrast, DNA damage inhibits SIRT activity, resulting in TopBP1 acetylation that constitutes DNA damage checkpoint and promote repair. SIRT1 pathway would deserve attention as a target for environmental contaminants (Figure 1). Furthermore, a downregulation of SIRT 1 may be induced through the activation of AhR, a crucial sensor of xenobiotics. It leads to a downregulation of peroxisome proliferator-activated receptor γ coactivator 1ߙ (PGC1ߙ) levels together with an increase of its acetylation (inactive fraction of PGC1 α ), followed by a decreased expression of the levels of phosphoenolpyruvate carboxykinase (PEPCK-C) and glucose-6-phosphate dehydrogenase (G6Pase) [6]. Altogether, these observations provide evidence of a role of SIRT1 in glucose homeostasis
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Impact of human monocyte and macrophage polarization on NLR expression and NLRP3 inflammasome activation

Impact of human monocyte and macrophage polarization on NLR expression and NLRP3 inflammasome activation

Innate response during inflammation is assured by the family of the evolutionarily con- served NLR receptors (Nucleotide-binding domain and Leucine-rich Repeat containing pro- teins) expressed mainly in monocytes and macrophages [ 8 ]. Upon detection of microbial (Pathogen-associated molecular patterns, PAMPs) or cell damage molecular patterns (Dam- age-associated molecular pattern, DAMPs), NLRs initiate the formation of multiprotein intra- cellular complexes called inflammasomes, which regulate secretion of the bioactive forms of the IL-1 cytokine family, through association with the adaptor protein ASC (Apoptosis-associ- ated Speck-like protein containing a Caspase recruitment domain), and activation of caspase-1 (encoded by CASP1) [ 9 – 11 ]. The secreted IL-1β and IL-18 play key roles in systemic inflam- mation promoting the expression of pro-inflammatory genes and thereby amplifying the inflammatory response. NLRs are multi-domain proteins. Their C-terminal domain is rich in leucine repeats (LRR), which under resting conditions, auto-inhibits the NLR [ 12 , 13 ]. The cen- tral nucleotide-binding domain (NACHT), mediates oligomerization and a variable N-termi- nal effector domain subdivides the NLR family into subgroups ( S1 Fig ) [ 14 ]. Among NLRs, NLRP1 [ 15 ], NLRP2 [ 16 ], NLRP3 [ 16 ], NLRP6 [ 17 ], NLRP7 [ 18 ], NLRP12 [ 19 ], NLRC4 and NLRB/NAIP [ 20 ] are considered as inflammasome-nucleating proteins. The NLRP3 inflam- masome gained a lot of attention due to the causality of NLRP3 mutations in cryopyrin-associ-
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Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability

Adaptation to DNA damage checkpoint in senescent telomerase-negative cells promotes genome instability

ilar results were found for tid1 Δ mutants (Fig. 3C–E). Thus, aberrant telomere homeostasis does not explain the lack of prolonged arrests in the adaptation mutants. We also asked whether the adaptation mutants might display defective checkpoint activation in response to telomeric damage. We considered this unlikely because while many proteins involved in adaptation also play roles in DNA damage repair and checkpoint activation, this is not the case for Cdc5 and Tid1 (Toczyski et al. 1997; Lee et al. 2001; Melo et al. 2001; Pellicioli et al. 2001; Harrison and Haber 2006; Clerici et al. 2014). Indeed, we specifically chose them to avoid confounding effects on checkpoint ac- tivation. Consistent with this, we found that cdc5-ad mu- tants and wild-type cells showed indistinguishable abilities to (1) form Ddc2-eGFP foci in the nucleus, (2) ar- rest in G2/M phase of the cell cycle, (3) undergo Rad53 hyperphosphorylation in response to DSBs induced by zeo- cin, and (4) undergo G2/M arrest at the restrictive temper- ature in a cdc13-1 background (in which telomeres become uncapped at high temperature, inducing checkpoint acti- vation) ( Supplemental Fig. S3A –D ; Supplemental Movie S2 ). Therefore, we conclude that the reduction in pro- longed nonterminal arrests during replicative senescence in the cdc5-ad mutant is not caused by disruption of DNA damage checkpoint activation.
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DNA damage and the activation of the p53 pathway mediate alterations in metabolic and secretory functions of adipocytes Running title: DNA damage and p53 in obese adipocytes

DNA damage and the activation of the p53 pathway mediate alterations in metabolic and secretory functions of adipocytes Running title: DNA damage and p53 in obese adipocytes

12 activity towards neutrophil, an effect which is similar or even greater (for doxorubicin treatment) than the neutrophil chemotactic factor fMLP (Fig. 4D). Microarray analysis of 3T3-L1 adipocytes treated with doxorubicin or nutlin-3 revealed that among the expressed chemokines, eleven were deregulated in one or both experimental conditions (Fig. 5A). Analysis by qRT-PCR confirmed that doxorubicin and nutlin-3 increased the mRNA expression of CCL9 (a fibroblast and dendritic cells chemotactic protein), CXCL1 ( a neutrophil chemotactic protein) and CCL2 while the expression of CCL7 mRNA, another monocytic chemotactic protein was increased by nutlin-3 only (Fig. 5B, C). Doxorubicin and nutlin-3 also induced the expression of IL-6. The silencing of p53 blocked the induction by nutlin-3 of all of these mRNAs (Fig.5B) and partly prevented their up- regulation in response to doxorubicin, except for CXCL1 (Fig. 5C). Since p53 silencing only partially reduced p53 expression and activation by doxorubicin (Supplementary Fig. 2C), we treated cells with both p53 siRNA and pifithrin-α, an inhibitor of p53 transcriptional activity. In this condition, CCL2/9, CXCL1, and IL-6 mRNA induction was prevented (Fig. 5C). Importantly, we found that the expression of mRNAs coding for CCL2/7/9 and CXCL1 was increased in epiAT from doxorubicin-treated, both in the adipocyte and SVF fractions for CCL2/7 and only in the adipocyte fraction for CCL9 and CXCL1 (Fig. 5D). We also examined the expression of these chemokines at the onset of obesity when DNA damage was observed in adipocytes. CCL2 mRNA level was increased after 2 weeks of HFD-feeding (Fig. 2I) and a trend toward an increase for CCL9 mRNA was observed (Fig. 5E). The mRNA levels of all of these chemokines were significantly increased after 4 weeks of HFD-feeding (Fig. 5F and Supplementary Fig. 1J).
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14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

Exactly what PTIP does to control the DNA damage response and coordinate DNA repair remains elusive. Much of the work performed on PTIP has focused on the interaction of PTIP with MLL2, MLL3, and MLL4 complexes [85] and the putative role of PTIP in transcription regulation. A recent study identified two PTIP containing complexes, a high molecular weight complex that co-purifies with MLL proteins, and a low molecular complex that purifies with PTIP-associated protein 1 (PA1). PA1 is recruited to IRIF by PTIP [83]. In addition, Gong and colleagues demonstrated that PTIP did not form foci in the absence of H2AX, MDC1 or RNF8. Interestingly, despite the in vitro and in vivo interaction between PTIP and 53BP1, PTIP and 53BP1 appear to be recruited to foci independently [83]. A number of important questions in the field remain unanswered, including the interplay between MCPH1 and MDC1, both of which seem to be interacting with γH2AX with similar affinities. It has been demonstrated that MCPH1 recruitment to foci does not depend on γ- H2AX; however, as MDC1 has been demonstrated to be a protein that coordinates and recruits several proteins to foci, the question remains, what is the role of MCPH1 in foci formation? Could MCPH1 be specifically involved in checkpoint exit? It has been demonstrated that MDC1 is degraded after DNA damage, suggesting that MCPH1 could bind to and facilitate the dephosphorylation of γH2AX after MDC1 destruction. It is also possible that MCPH1 regulates repair processes like HR, while MDC1 might functions primarily in the early stages of the DNA damage signaling response. Another facet of phosphopeptide binding in protein recruitment to foci after DNA damage is the question of uniqueness in ligand binding. It is not intuitive that recruitment of phosphopeptide binding proteins into nuclear foci should depend exclusively on a single ligand. Given the large number of ATM substrates, why should proteins like BRCA1 bind to such a limited number of ligands after DNA damage, especially when 14-3-3 proteins typically bind hundreds of distinct ligands. It seems likely that there are other factors involved in specifically localizing proteins to foci.
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Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis

Role of the Polymerase ϵ sub-unit DPB2 in DNA replication, cell cycle regulation and DNA damage response in Arabidopsis

Genetic analysis reveals complex interactions of DPB2 func- tion with DDR The tolerance to different types of DNA damage that trig- ger replicative stress or DNA breaks, may be due to consti- tutive activation of ATR, and /or ATM. To investigate the contribution of the DNA damage response to the growth defects caused by DPB2 over-expression, the DPB2OE con- struct was introduced in atm, atr, and sog1 mutants. For each background, two transformation batches were anal- ysed and at least 63 plants of each T1 generation were grown in the greenhouse. We were able to identify plants display- ing a clear DPB2OE phenotype in the T1 generation in all backgrounds (Table 3 , Supplementary Figure S10A–C), but the distribution of plants in the different phenotypic cate- gories was significantly different from what was observed in the wild-type in all genotypes ( χ 2 , P-value < 0.01). DPB2 over-expression was quantified using qRT-PCR in different lines representative of the different phenotypic groups (Sup- plementary Figure S10D). Interestingly, plants with similar phenotypes in the Col-0, atm or sog1 background had sim- ilar DPB2 expression levels. By contrast, in the atr back- ground, much higher DPB2 over-expression was required to induce severe and intermediate developmental defects, in- dicating that ATR activation accounts for some of the phe- notypic alterations induced by DPB2 over-expression. Root growth assays confirmed that plants had been assigned to the proper phenotypic category: root growth inhibition was similar in the severe, intermediate and mild lines of all tested backgrounds (Supplementary Figure S11). Interestingly, vi- able plants with very severe phenotype could only be ob- tained in the sog1, and atr backgrounds (Supplementary Figure S10A and B), but many of these plants died before flowering, and they were obtained at a lower frequency than in the Col-0 background (Table 3 ). In the atm background, we only identified plants with intermediate and mild vege- tative phenotype (Supplementary Figure S10C). These re- sults suggest that ATM is required for survival of severe
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Cell-Based Biosensor to Report DNA Damage in Micro- and Nanosystems

Cell-Based Biosensor to Report DNA Damage in Micro- and Nanosystems

Here we introduced an open-source biosensor speci fically engineered to report on DNA damage induced by micro- and nanosystems. This transcriptional sensor reports on activation of p21, a crucial and well-characterized node in the DNA damage and cell cycle arrest pathway, via p53-driven TurboRFP protein expression. The biosensor allows visual and non- destructive assessment of gene expression with single-cell resolution using commonly available equipment to quantify the cellular fluorescence response without requiring additional reagents and materials, large numbers of cells, or overly sophisticated microscopy. We engineered the biosensor cells to use TurboRFP because of its intracellular stability, which allows RFP to be assayed over a range of times and because its spectral characteristics allows it to be more-easily multiplexed with other assays than a GFP-based reporter. We chose NIH-3T3 cells as the background because they are one of the most commonly used fibroblast cell lines, are very easy to culture, express wild type p53 protein, and have been extensively studied for DNA damage, 48,49 showing sensitivity to different types of genotoxic agents. We have also deposited the reporter plasmid in Addgene for users wishing to create the sensor in di fferent background cells to get specific stress responses, while our biosensor is available from us upon request. After validating that expression of TurboRFP was dependent on the p53 availability, we characterized our biosensor using agents with known genotoxic function. The biosensor cells were exposed to a range of concentrations, from very low concentration that did not induce signi ficant fluorescence responses up to concen- trations that were cytotoxic and caused cell death, in order to determine the dynamics of the dose- and time-response. Most genotoxic agents at high enough concentration induced a signi ficant response of the biosensor 12 h after exposure, including MMS, UV-C, hydrogen peroxide, and Ag-NPs, but at this time point it was not always possible to distinguish between responses to di fferent doses or concentrations. At exposure times of ≥24 h it was possible to detect the fold- induction of red fluorescence caused by lower but nevertheless genotoxic concentrations of the stressors. Thus, it is likely that for most applications read-out at ∼24 h following exposure will be optimal. This read-out time is similar to those of commercially available cell-based assays, which also range between 24 and 48 h. The sensitivity of our biosensor for MMS genotoxicity detection is similar to that of the GreenScreen assay. 33
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Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

MERIT40 was shown to be required for the integrity of the BRCA1A complex containing RAP80, BRCA1, BRCC36 and CCDC98 [ 24 ] [ 25 ]. We thus tested if other subunits of this com- plex are also loaded to chromatin bound by TNKS1. We indeed found that RAP80 was recruited to the lacO array with 100% efficiency in a PARP activity independent manner by TNKS 1 ( Fig 8A ). To verify that TNKS1 tethering does not trigger the DDR, we also observed γ-H2AX, MDC1 and 53BP1 accumulation at the TNKS1 bound array, but none of these factors or modifications could be detected ( S7A Fig ). To test the potential role of TNKSs in recruiting BRCA1A subunits to chromatin after DNA damage, we depleted TNKS1 and 2 in U2OS17 cells and tested the efficiency of MERIT40 and RAP80 recruitment to ISce-I induced DSBs ( Fig 8B ). Compared to the scrambled control, downregulation of TNKS expression decreased the association of all these factors with DSBs ( Fig 8B ). On the other hand, downregulation of lacR, FN-TNKS1 and ISce-I. 20hours before fixation, cells were treated with mimosine to block the cell cycle in G1. Percent of cells with FN-TNKS1 signal on the lacO array was determined. Results from three independent experiments are shown with SD (N = 100). (C) TNKS1 is recruited to DSBs in vivo in a subset of cells. U2OS cells were transfected with FN-TNKS1, treated with NCS and immunostained with antibodies against flag and γ-H2AX. Cells were blocked in G2 phase by the CDK1 inhibitor RO-3306 on panels e-g. Frequency of cells showing a colocalizing signal for γ-H2AX and FN-TNKS increased from 24% (asynchronous cells) to 70% (G2 cells) as shown on the graph on the right. Colocalization was analyzed by plotting the intensity of the red/green signal against the distance along the white line on the merged image. The starting point is marked by a star. (D) MDC1 is required for TNKS recruitment to pure DSBs in vivo. U2OS17 cells were transfected with control or MDC1 targeted siRNAs. 24hours after siRNA treatment, cells were co-transfected with mCherry- lacR, FN-TNKS1 or MN-TNKS2 and ISce-I. The frequency of cells having TNKS1/2 signal on the array was determined, results of three independent experiments are shown with SD (N = 100). (E) MDC1 is required for TNKS foci formation. U2OS cells were transfected with control or MDC1-directed siRNAs. 48 hours later the cells were transfected with FN-TNKS1 and 24 hours later treated with 100ng/ml NCS. 6 hours after treatment cells were stained with antibodies against γ-H2AX and FN-TNKS. Representative confocal microscopy pictures are shown.
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Evaluating DNA damage response (DDR) activation in human prostate cancer

Evaluating DNA damage response (DDR) activation in human prostate cancer

5.0 Conclusion   DDR is a complex signalling network and its failure causes genomic instability, an underlying cause of cancer. Today, the tumor’s biology in individual prostate cancer patient is still not taken into consideration for clinical management of the disease. In an era of evolving personalized medicine, we confirmed that RELA (p65) and showed that DDR marker 53BP1 have prognostic value in patients with prostate adenocarcinoma. Patients expressing reduced amounts of 53BP1 have a better bone metastasis free survival (p=0.005) and those with reduced amounts of RELA have a better biochemical control (p=0.002). These findings were also correlated in univariate and multivariate cox regression analysis. Even though our follow up is quite long (101.5 months), a longer follow-up is probably needed to translate into a correlation with survival since prostate cancer patients have very good prognosis and develop metastasis leading to death many years after the initial curative treatment. Future work should focus on designing the ways to better predict responses of individual patients to DNA damaging therapies including radiation and various chemotherapeutics that are used in first line of treatment. These predictions could be based on the genetic and functional profiling of patient specific tumors. This may facilitate selection of a proper modality or combination of treatment options on an individualized basis and also optimize the dosage of such therapies according to the state of the DNA damage checkpoint and repair machineries of each individual patient.
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Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation

Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation

1 Institut Pasteur, Unite´ Plasticite´ du Ge´nome Bacte´rien, De´partement Ge´nomes et Ge´ne´tique, Paris, France, 2 CNRS, URA2171, Paris, France Abstract Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.
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Uropathogenic E. coli induces DNA damage in the bladder

Uropathogenic E. coli induces DNA damage in the bladder

The role of pks island in UPEC cannot merely be reduced to a determinant of intestinal col- onisation. Indeed, our current study shows that the pks machinery is also active in the urinary tract and even within IBCs. Bacterial multiplication in these structures is intense, with a dou- bling time of nearly 30 min, which requires an efficient and optimised bacterial metabolism [ 35 ]. Although colibactin is a small molecule, the metabolic cost of its production, i.e. express- ing and operating the PK-NRP biosynthesis machinery is very high: nearly 1000 times higher than that of peptide synthesis [ 36 ]. As UPEC trigger such energetically inexpedient assembly lines during UTIs, one may speculate that they must derive an adaptive benefit from it. Other products of the machinery may possibly confer this advantage. Indeed, there is a wide diversity of metabolites produced by the pks machinery (at least in vitro), ranging from other putative forms of “colibactins” (such as macrocyclic metabolites) to other smaller metabolites, which potentially vary between strains [ 15 , 37 ]. The biological function of these metabolites remains to be elucidated, but some of these metabolites may be relevant to the pathogenesis of infec- tions. We have, for instance, recently described the synthesis of C12-Asn-GABA, by the pks machinery of the probiotic Nissle 1917 strain and shown its digestive pain-relieving activity [ 38 ]. The production of such an analgesic metabolite coupled with the increased production of siderophores by pks+ E. coli strains may provide a selective advantage to colonising the urinary tract, in addition to the digestive tract. Future experiments should aim at examining the impact of the various pks-associated factors on the course of the infection.
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Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

Comet assay on thawed embryos: An optimized technique to evaluate DNA damage in mouse embryos

The main technical details of the published protocols are presented in Table 2 . We selected 14 studies using the comet assay on animal embryos matching our criteria, out of 118 studies retrieved. In these studies, fresh embryos were all obtained by natural mating ( Fabian et al., 2003; Harrouk et al., 2000; Müller et al., 1996; Takahashi et al., 1999; Tranguch et al., 2003; Webster et al., 2000 ) or IVF ( Hwang et al., 2013; Ju et al., 2010; Kitagawa et al., 2004; Natarajan et al., 2010; Rajesh et al., 2010; Sturmey et al., 2009; Takahashi et al., 2000; Thiyagarajan and Valivittan, 2009a, 2009b ). This review of the litera- ture shows that there is no standardization of the protocol: Seven dif- ferent animal species were used; The ZP was removed in 4 studies; The protocols for the transfer of the embryos in the LMP agarose layer were heterogeneous: a minimum of half a blastocyst and a maximum of 20 cleavage embryos were transferred, and the volume of LMP varied from 4 μL to 50 μL; The lysis and electrophoresis protocols were also het- erogeneous. No publication described the recovery rate of the embryos in the agarose layer after lysis and electrophoresis.
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Damage response of sandwich plates subject to dynamic loads

Damage response of sandwich plates subject to dynamic loads

The analytical predictions for the central deflection of the plate when subjected to low velocity impact loads are within 5% error for the fully-plastic, isotropic facesheet and 1[r]

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Electrical discharges in water induce spores' DNA damage

Electrical discharges in water induce spores' DNA damage

fragments were much more degraded (lane 12) with a loss of intensity of 93%. Thus, the pres- ence of pyrimidine dimers was exhibited after electric arcs exposure. Discussion After electric arcs exposure, morphological imaging by SEM did not reveal any variation of spore size and the protein ridges remained intact on the coat surface. AFM in liquid validated these observations at the nanoscale and no variation of roughness was observed. TEM images confirmed the preservation of the spore structure and the visible integrity of the spores. These results demonstrated a non-physical destruction of spores at the nanometric scale by the elec- tric arcs. In the literature, the shock waves are currently described as an efficient method to inactivate bacteria [ 37 , 38 ] by a mechanical disruption of the bacterial cell wall [ 14 ]. Further- more, in a previous work we demonstrated that the disorganization of the spore structure is caused by Pulsed Electric Fields (PEF) [ 21 ]. Here, the absence of visible damage on the spore structure, suggested the non-implication of PEF during electric arcs exposure. In conclusion, the inactivation of spores during electric arcs treatment was not due to electric fields nor to shock waves. However, the possible presence of cell wall damage in vegetative bacteria sug- gested that more vulnerable microorganisms than spores could be impacted by electric fields and shock waves.
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Uropathogenic E. coli induces DNA damage in the bladder

Uropathogenic E. coli induces DNA damage in the bladder

cells, including in urothelial regenerative cells and that colibactin is produced by pks+ UPEC.. 618[r]

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A Chromatin-Dependent Role of the Fragile X Mental Retardation Protein FMRP in the DNA Damage Response

A Chromatin-Dependent Role of the Fragile X Mental Retardation Protein FMRP in the DNA Damage Response

To confirm that FMRP KO MEFs are defective in their response to replication stress, we subjected FMRP KO MEFs to additional sources of replication stress agents including hydroxyurea (HU) and UV irradiation. In both cases, FMRP KO MEFs failed to show a time-dependent increase of the γH2A.X level as compared to wild type MEFs (10-fold induction at 60 min post-treatment) (Fig. 1C, compare lanes 1–4 with 5–8 of the upper and lower panels). Importantly, FMRP KO MEFs reconstituted with a FLAG-HA epitope- tagged, wild type FMRP (Flag-HA-FMRP), conferred a more robust γH2A.X response to increasing concentrations of APH compared with the Flag-HA vector alone (Fig. 1D and Fig. S1D) (12-fold induction in Flag-HA-FMRP cells as compared to 4-fold induction in Flag-HA only cells). This was not a MEF-cell-specific effect since reduction of FMRP in HeLa cells by RNAi also resulted in a compromised induction of γH2AX in response to replication stress (Fig. 1E). In addition to H2A.X phosphorylation regulation, loss of FMRP also affected another ATR-dependent, replication response-specific phosphorylation event, phosphorylation of BRCA1 at Ser-1423 (Tibbetts et al., 2000) (Fig. S1E, F). Consistent with the potential role of FMRP in the replication stress response, FMRP RNAi knockdown HeLa cells reconstituted with Flag-HA vector alone, but not tagged wild type FMRP (Flag- HA-FMRP) were more sensitive to replication stress in the clonogenic survival assay (Fig. S2A, B), and FMRP KO MEFs were also more sensitive to replication stress compared to wild type MEFs (Fig. S2C). These findings are in line with previous reports describing a pro-survival role of FMRP (Jeon et al., 2012; Jeon et al., 2011; Liu et al., 2012). Taken together, the above findings link FMRP to replication stress-induced DNA damage response and indicate that FMRP may be part of the ATR-dependent signaling pathway.
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The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage

The transcription-coupled DNA repair-initiating protein CSB promotes XRCC1 recruitment to oxidative DNA damage

Received October 20, 2017; Revised June 07, 2018; Editorial Decision June 13, 2018; Accepted June 22, 2018 ABSTRACT Transcription-coupled nucleotide excision repair fac- tor Cockayne syndrome protein B (CSB) was sug- gested to function in the repair of oxidative DNA damage. However thus far, no clear role for CSB in base excision repair (BER), the dedicated path- way to remove abundant oxidative DNA damage, could be established. Using live cell imaging with a laser-assisted procedure to locally induce 8-oxo- 7,8-dihydroguanine (8-oxoG) lesions, we previously showed that CSB is recruited to these lesions in a transcription-dependent but NER-independent fash- ion. Here we showed that recruitment of the pre- ferred 8-oxoG-glycosylase 1 (OGG1) is independent of CSB or active transcription. In contrast, recruit- ment of the BER-scaffolding protein, X-ray repair cross-complementing protein 1 (XRCC1), to 8-oxoG lesions is stimulated by CSB and transcription. Re- markably, recruitment of XRCC1 to BER-unrelated single strand breaks (SSBs) does not require CSB or transcription. Together, our results suggest a spe- cific transcription-dependent role for CSB in recruit- ing XRCC1 to BER-generated SSBs, whereas XRCC1 recruitment to SSBs generated independently of BER relies predominantly on PARP activation. Based on our results, we propose a model in which CSB plays a role in facilitating BER progression at transcribed
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