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The Journal of Cell Biology Aug 2021Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost...
Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress-response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle-TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle-TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.
Topics: Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Carrier Proteins; Cell Nucleolus; DNA Damage; DNA Replication; DNA, Ribosomal; DNA-Binding Proteins; Genomic Instability; HCT116 Cells; HeLa Cells; Humans; Hydroxyurea; Microscopy, Fluorescence; Nuclear Proteins; Phosphoproteins; Protein Binding; Protein Transport; Signal Transduction
PubMed: 34100862
DOI: 10.1083/jcb.202008085 -
Chromosome Research : An International... Aug 2023Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural...
Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.
Topics: Humans; Induced Pluripotent Stem Cells; Aphidicolin; Caffeine; Chromosomes, Human, Pair 12; In Situ Hybridization, Fluorescence; Intracellular Signaling Peptides and Proteins
PubMed: 37597021
DOI: 10.1007/s10577-023-09729-5 -
Nature Communications Apr 2021The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around...
The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.
Topics: Aphidicolin; Calreticulin; Clonal Hematopoiesis; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Humans; Leukemia, Myeloid; Myeloid Progenitor Cells; Poly (ADP-Ribose) Polymerase-1; Repressor Proteins; Sequence Deletion; Serine-Arginine Splicing Factors; DNA Polymerase theta
PubMed: 33911081
DOI: 10.1038/s41467-021-22803-y -
Life Sciences Nov 2023Studies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments,...
AIMS
Studies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments, however, clinical use of ATR inhibitors in combination with DNA damaging chemotherapy is limited due to toxicity in healthy tissues. In this study, we investigated the synergistic anticancer effect between ATR inhibition and oxidative DNA damage induced by the thioredoxin reductase inhibitor auranofin.
MAIN METHODS
Cytotoxicity was evaluated by cell viability assays. Western blot, comet assay, immunostaining and flow cytometry were performed to dissect the underlying mechanisms. In vivo efficacy was examined against tumor xenografts.
KEY FINDINGS
Nontoxic doses of auranofin alone increased the levels of reactive oxygen species (ROS) in cancer but not noncancerous cells, resulting in oxidative DNA damage and activation of the ATR DNA damage response pathway selectively in cancer cells. Inhibition of ATR in auranofin-treated cancer cells resulted in unscheduled firing of dormant DNA replication origins, abrogation of the S phase cell cycle checkpoint and extensive DNA breakage, leading to replication catastrophe and potent synergistic lethality. Both the antioxidant NAC and the DNA polymerase inhibitor aphidicolin reduced replication stress and synergistic cytotoxicity, implicating replication stress-driven catastrophic cell death resulted from collision between oxidative DNA damage and dysregulated DNA replication. In vivo, auranofin and VE822 coadministration enabled marked regressions of tumor xenografts, while each drug alone had no effect.
SIGNIFICANCE
As increased generation of ROS is a universal feature of tumors, our findings may open new routes to broaden the therapeutic potential of ATR inhibitors.
Topics: Humans; Auranofin; Reactive Oxygen Species; DNA Damage; Neoplasms; Oxidative Stress; Protein Kinase Inhibitors; DNA; Checkpoint Kinase 1; Cell Line, Tumor; Ataxia Telangiectasia Mutated Proteins
PubMed: 37778414
DOI: 10.1016/j.lfs.2023.122131 -
FEBS Letters Mar 2021We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous...
We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between the FA protein FANCD2 and the non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in the FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediate stalled replication fork restart, and prevent nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability.
Topics: Aphidicolin; Cell Survival; DNA; DNA Repair; DNA Replication; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Fibroblasts; Genome, Human; Genomic Instability; Humans; Hydroxyurea; Mitomycin; Primary Cell Culture; Protein Binding; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases
PubMed: 33423298
DOI: 10.1002/1873-3468.14035 -
Analytical Biochemistry Feb 2021The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is...
The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.
Topics: Bromodeoxyuridine; Carbocyanines; Cell Cycle; DNA; DNA Replication; Flow Cytometry; Fluorescent Dyes; HL-60 Cells; HeLa Cells; Humans; Jurkat Cells; Reproducibility of Results; Staining and Labeling
PubMed: 33159846
DOI: 10.1016/j.ab.2020.114002 -
Viruses Oct 2020Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal...
Identification of a New Equid Herpesvirus 1 DNA Polymerase (ORF30) Genotype with the Isolation of a C/H Strain in French Horses Showing no Major Impact on the Strain Behaviour.
Equid herpesvirus 1 is one of the most common viral pathogens in the horse population and is associated with respiratory disease, abortion and still-birth, neonatal death and neurological disease. A single point mutation in the DNA polymerase gene (ORF30: A2254G, N752D) has been widely associated with neuropathogenicity of strains, although this association has not been exclusive. This study describes the fortuitous isolation of a strain carrying a new genotype C (H) from an outbreak in France that lasted several weeks in 2018 and involved 82 horses, two of which showed neurological signs of disease. The strain was characterised as U clade 10 using the equid herpesvirus 1 (EHV-1) multi-locus sequence typing (MLST) classification but has not been identified or isolated since 2018. The retrospective screening of EHV-1 strains collected between 2016 and 2018 did not reveal the presence of the C mutation. When cultured in vitro, the C EHV-1 strain induced a typical EHV-1 syncytium and cytopathic effect but no significant difference was observed when compared with A and G EHV-1 strains. An experimental infection was carried out on four Welsh mountain ponies to confirm the infectious nature of the C strain. A rapid onset of marked respiratory disease lasting at least 2 weeks, with significant virus shedding and cell-associated viraemia, was observed. Finally, an in vitro antiviral assay using impedance measurement and viral load quantification was performed with three antiviral molecules (ganciclovir (GCV), aciclovir (ACV) and aphidicolin (APD)) on the newly isolated C strain and two other A/G field strains. The three strains showed similar sensitivity to ganciclovir and aphidicolin but both C and A strains were more sensitive to aciclovir than the G strain, based on viral load measurement.
Topics: Animals; DNA-Directed DNA Polymerase; Disease Outbreaks; France; Genotype; Herpesviridae Infections; Herpesvirus 1, Equid; Horse Diseases; Horses; Male; Mutation; Open Reading Frames; Retrospective Studies; Viral Load; Viral Proteins
PubMed: 33066315
DOI: 10.3390/v12101160 -
Cell Reports Apr 2022Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild...
Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS.
Topics: DNA; DNA Replication; Mitosis
PubMed: 35443178
DOI: 10.1016/j.celrep.2022.110701 -
DNA Repair Feb 2023New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary...
New development and optimization of oncologic strategies are steadily increasing the number of long-term cancer survivors being at risk of developing second primary neoplasms (SPNs) as a late consequence of genotoxic cancer therapies with the highest risk among former childhood cancer patients. Since risk factors and predictive biomarkers for therapy-associated SPN remain unknown, we examined the sensitivity to mild replication stress as a driver of genomic instability and carcinogenesis in fibroblasts from 23 long-term survivors of a pediatric first primary neoplasm (FPN), 22 patients with the same FPN and a subsequent SPN, and 22 controls with no neoplasm (NN) using the cytokinesis-block micronucleus (CBMN) assay. Mild replication stress was induced with the DNA-polymerase inhibitor aphidicolin (APH). Fibroblasts from patients with the DNA repair deficiency syndromes Bloom, Seckel, and Fanconi anemia served as positive controls and for validation of the CBMN assay supplemented by analysis of chromosomal aberrations, DNA repair foci (γH2AX/53BP1), and cell cycle regulation. APH treatment resulted in G2/M arrest and underestimation of cytogenetic damage beyond G2, which could be overcome by inhibition of Chk1. Basal micronuclei were significantly increased in DNA repair deficiency syndromes but comparable between NN, FPN, and SPN donors. After APH-induced replication stress, the average yield of micronuclei was significantly elevated in SPN donors compared to FPN (p = 0.013) as well as NN (p = 0.03) donors but substantially lower than for DNA repair deficiency syndromes. Our findings suggest that mild impairment of the response to replication stress induced by genotoxic impacts of DNA-damaging cancer therapies promotes genomic instability in a subset of long-term cancer survivors and may drive the development of an SPN. Our study provides a basis for detailed mechanistic studies as well as predictive bioassays for clinical surveillance, to identify cancer patients at high risk for SPNs at first diagnosis.
Topics: Humans; Child; Cancer Survivors; Neoplasms, Second Primary; Apoptosis; Cell Line, Tumor; G2 Phase Cell Cycle Checkpoints; Chromosomal Instability; Genomic Instability; Micronucleus Tests; DNA Damage; DNA; Fibroblasts
PubMed: 36549044
DOI: 10.1016/j.dnarep.2022.103435 -
STAR Protocols Mar 2023Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we...
Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. We use EdU incorporation to label nascent DNA in mitotic cells, followed by isolation of the EdU-labeled DNA and next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Groelly et al. (2022) and Macheret et al. (2020)..
Topics: DNA Replication; DNA; Cells, Cultured; Mitosis; DNA Repair
PubMed: 36598851
DOI: 10.1016/j.xpro.2022.101970