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Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites’ cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.

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Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.

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ARID1A, an epigenetic tumor suppressor, is the most common gene mutation in clear-cell ovarian cancers (CCOCs). CCOCs are often resistant to standard chemotherapy and lack effective therapies. We hypothesized that ARID1A loss would increase CCOC cell dependency on chromatin remodeling and DNA repair pathways for survival. We demonstrate that combining BRD4 inhibitor (BRD4i) with DNA damage response inhibitors (ATR or WEE1 inhibitors; e.g. BRD4i-ATRi) was synergistic at low doses leading to decreased survival, and colony formation in CCOC in an ARID1A dependent manner. BRD4i-ATRi caused significant tumor regression and increased overall survival in ARID1AMUT but not ARID1AWT patient-derived xenografts. Combination BRD4i-ATRi significantly increased γH2AX, and decreased RAD51 foci and BRCA1 expression, suggesting decreased ability to repair DNA double-strand-breaks (DSBs) by homologous-recombination in ARID1AMUT cells, and these effects were greater than monotherapies. These studies demonstrate BRD4i-ATRi is an effective treatment strategy that capitalizes on synthetic lethality with ARID1A loss in CCOC.

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Mycobacterium bovis BCG Moreau is the main Brazilian strain for vaccination against tuberculosis. It is considered an early strain, more like the original BCG, whereas BCG Pasteur, largely used as a reference, belongs to the late strain clade. BCG Moreau, contrary to Pasteur, is naturally deficient in homologous recombination (HR). In this work, using a UV exposure test, we aimed to detect differences in the survival of various BCG strains after DNA damage. Transcription of core and regulatory HR genes was further analyzed using RT-qPCR, aiming to identify the molecular agent responsible for this phenotype. We show that early strains share the Moreau low survival rate after UV exposure, whereas late strains mimic the Pasteur phenotype, indicating that this increase in HR efficiency is linked to the evolutionary clade history. Additionally, RT-qPCR shows that BCG Moreau has an overall lower level of these transcripts than Pasteur, indicating a correlation between this gene expression profile and HR efficiency. Further assays should be performed to fully identify the molecular mechanism that may explain this differential phenotype between early and late BCG strains.

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DNA damage response (DDR) pathways in keto-enol genotoxicity have not been characterized, and few studies have reported genotoxic effects in non-target organisms. The present study shows that concentrations of 11.2, 22.4, 37.3 mg/L of Movento® 240SC and 12.3, 24.6, 41.1 mg/L of Envidor® 240SC for 72 h oral exposure induced DSBs by significantly increasing the percentage of γH2AV expression in regions 2b and 3 from the germarium of wild type females of Drosophila melanogaster Oregon R, compared to the control group (0.0 mg/L of insecticides), via confocal immunofluorescence microscopy. The comparison between both insecticides' reveals that only the Envidor® 240SC induces concentration-dependent DNA damage, as well as structural changes in the germarium. We determined that the DDR induced by Movento® 240SC depends on the activation of the ATMtefu, Chk1grp and Chk2lok kinases by significantly increasing the percentage of expression of γH2AV in regions 2b and 3 of the germarium, and that ATRmei-29D and p53dp53 kinases only respond at the highest concentration of 37.3 mg/L of Movento® 240SC. With the Envidor® 240SC insecticide, we determined that the DDR depends on the activation of the ATRmei-29D/Chk1grp and ATMtefu/Chk2lok kinases, and p53dp53 by significantly increasing the percentage of expression of γH2AV in the germarium.

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In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.

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Organisms have evolved a complex system, called the DNA damage response (DDR), which maintains genome integrity. The DDR is responsible for identifying and repairing a variety of lesions and alterations in DNA. DDR proteins coordinate DNA damage detection, cell cycle arrest, and repair, with many of these events regulated by protein phosphorylation. In the human proteome, 23 proteins contain the BRCT (BRCA1 C-Terminus domain) domain, a modular signaling domain that can bind phosphopeptides and mediate protein-protein interactions. BRCTs can be found as functional single units, tandem (tBRCT), triplet (tpBRCT), and quartet. Here we examine the evolution of the tpBRCT architecture present in TOPBP1 (DNA topoisomerase II binding protein 1) and ECT2 (epithelial cell transforming 2), and their respective interaction partners RAD9 (Cell cycle checkpoint control protein RAD9) and CYK-4 (Rac GTPase-activating protein 1), with a focus on the conservation of the phosphopeptide-binding residues. The pair TOPBP1-RAD9 arose with the Eukaryotes and ECT2-CYK-4 with the Eumetazoans. Triplet structural and functional characteristics were conserved in almost all organisms. The first unit of the triplet (BRCT0) is different from the other two BRCTs but conserved between orthologs for both TOPBP1 and ECT2. BRCT domain evolution simulations suggest a trend to retain the singlet or towards two or three BRCT copies per protein consistent with functional tBRCT and tpBRCT architectures. Our results shed light on the emergence of the function and architecture of multiple BRCT domain organizations and provide information about the evolution of the BRCT triplet. Knowledge of BRCT domain evolution can improve the understanding of DNA damage response mechanisms and signal transduction in DDR.

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Introduction: Metastatic breast cancer causes the most breast cancer-related deaths around the world, especially in countries where breast cancer is detected late into its development. Genetic testing for cancer susceptibility started with the BRCA 1 and 2 genes. Still, recent research has shown that variations in other members of the DNA damage response (DDR) are also associated with elevated cancer risk, opening new opportunities for enhanced genetic testing strategies. Methods: We sequenced BRCA1/2 and twelve other DDR genes from a Mexican-mestizo population of 40 metastatic breast cancer patients through semiconductor sequencing. Results: Overall, we found 22 variants -9 of them reported for the first time- and a strikingly high proportion of variations in ARID1A. The presence of at least one variant in the ARID1A, BRCA1, BRCA2, or FANCA genes was associated with worse progression-free survival and overall survival in our patient cohort. Discussion: Our results reflected the unique characteristics of the Mexican-mestizo population as the proportion of variants we found differed from that of other global populations. Based on these findings, we suggest routine screening for variants in ARID1A along with BRCA1/2 in breast cancer patients from the Mexican-mestizo population.

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Cell fate determination is a complex process that is frequently described as cells traveling on rugged pathways, beginning with DNA damage response (DDR). Tumor protein p53 (p53) and phosphatase and tensin homolog (PTEN) are two critical players in this process. Although both of these proteins are known to be key cell fate regulators, the exact mechanism by which they collaborate in the DDR remains unknown. Thus, we propose a dynamic Boolean network. Our model incorporates experimental data obtained from NSCLC cells and is the first of its kind. Our network's wild-type system shows that DDR activates the G2/M checkpoint, and this triggers a cascade of events, involving p53 and PTEN, that ultimately lead to the four potential phenotypes: cell cycle arrest, senescence, autophagy, and apoptosis (quadra-stable dynamics). The network predictions correspond with the gain-and-loss of function investigations in the additional two cell lines (HeLa and MCF-7). Our findings imply that p53 and PTEN act as molecular switches that activate or deactivate specific pathways to govern cell fate decisions. Thus, our network facilitates the direct investigation of quadruplicate cell fate decisions in DDR. Therefore, we concluded that concurrently controlling PTEN and p53 dynamics may be a viable strategy for enhancing clinical outcomes.

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BACKGROUND: Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. METHODS: Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. RESULTS: VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. CONCLUSIONS: VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

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The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

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RAS oncogenes are chief tumorigenic drivers, and their mutation constitutes a universal predictor of poor outcome and treatment resistance. Despite more than 30 years of intensive research since the identification of the first RAS mutation, most attempts to therapeutically target RAS mutants have failed to reach the clinic. In fact, the first mutant RAS inhibitor, Sotorasib, was only approved by the FDA until 2021. However, since Sotorasib targets the KRAS G12C mutant with high specificity, relatively few patients will benefit from this therapy. On the other hand, indirect approaches to inhibit the RAS pathway have revealed very intricate cascades involving feedback loops impossible to overcome with currently available therapies. Some of these mechanisms play different roles along the multistep carcinogenic process. For instance, although mutant RAS increases replicative, metabolic and oxidative stress, adaptive responses alleviate these conditions to preserve cellular survival and avoid the onset of oncogene-induced senescence during tumorigenesis. The resulting rewiring of cellular mechanisms involves the DNA damage response and pathways associated with oxidative stress, which are co-opted by cancer cells to promote survival, proliferation, and chemo- and radioresistance. Nonetheless, these systems become so crucial to cancer cells that they can be exploited as specific tumor vulnerabilities. Here, we discuss key aspects of RAS biology and detail some of the mechanisms that mediate chemo- and radiotherapy resistance of mutant RAS cancers through the DNA repair pathways. We also discuss recent progress in therapeutic RAS targeting and propose future directions for the field.

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PIMREG expression strongly correlates with cellular proliferation in both malignant and normal cells. Throughout embryo development, PIMREG expression is prominent in the central nervous system. Recent studies have described elevated PIMREG expression in different types of tumors, which correlates with patient survival and tumor aggressiveness. Given the emerging significance of PIMREG in carcinogenesis and its putative role in the context of the nervous system, we investigated the expression and function of PIMREG in gliomas, the most common primary brain tumors. We performed an extensive analysis of PIMREG expression in tumors samples from glioma patients. We then assessed the effects of PIMREG silencing and overexpression on the sensitivity of glioblastoma cell lines treated with genotoxic agents commonly used for treating patients and assessed for treatment response, proliferation and migration. Our analysis shows that glioblastoma exhibits the highest levels of PIMREG expression among all cancers analyzed and that elevated PIMREG expression is a biomarker for glioma progression and patient outcome. Moreover, PIMREG is induced by genotoxic agents, and its silencing renders glioblastoma cells sensitive to temozolomide treatment and affects ATR- and ATM-dependent signaling. Our data demonstrate that PIMREG is involved in DNA damage response and temozolomide resistance of glioblastoma cells and further supports a role for PIMREG in tumorigenesis.

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Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the nucleotide excision DNA repair system, but this function in plants has not been investigated. We studied the role of MED17 in Arabidopsis plants exposed to UV-B radiation. Our results demonstrate that med17 and OE MED17 plants have altered responses to UV-B, and that MED17 participates in various aspects of the DNA damage response (DDR). Comparison of the med17 transcriptome with that of wild-type (WT) plants showed that almost one-third of transcripts with altered expression in med17 plants were also changed by UV-B exposure in WT plants. Increased sensitivity to DNA damage after UV-B in med17 plants could result from the altered regulation of UV-B responsive transcripts but MED17 also physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we show that MED17 is necessary to regulate the DDR activated by ataxia telangiectasia and Rad3 related (ATR), and that programmed cell death 5 (PDCD5) overexpression reverts the deficiencies in DDR shown in med17 mutants. Our data demonstrate that MED17 is an important regulator of DDR after UV-B irradiation in Arabidopsis.

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We previously showed that farmers in Bolivia are exposed to many pesticides, some at elevated levels, and that this was associated with increased risk of genetic damage. To improve the understanding of possible mixture effects, the cytotoxicity and genotoxicity of pesticides were studied in vitro using human liver HepG2 cells. The studied pesticides were 2,4-D, chlorpyrifos, cypermethrin, glyphosate, methamidophos, paraquat, profenofos, and tebuconazole. Three mixtures (U1, U2, and U3) were based on profiles of urinary pesticide metabolites and one mixture on the most frequently used pesticides (S1). The results showed that paraquat and methamidophos were the most cytotoxic pesticides (EC50 ≤0.3 mM). Paraquat, chlorpyrifos, tebuconazole, and the U1, U2, and U3 mixtures, which contained a large proportion of either chlorpyrifos or tebuconazole, significantly increased intracellular ROS levels. Most pesticides activated DNA damage signaling through proteins Chk1 and H2AX. Strongest responses were elicited by paraquat, profenofos, chlorpyrifos, cypermethrin, and the S1 mixture, which contained 25% paraquat. Comet assay revealed significant increases of DNA damage in response to paraquat, cypermethrin, and U2 and S1 mixtures, which contained high levels of cypermethrin and paraquat, respectively. In summary, we showed that the tested pesticides, alone or in mixtures, in general induced oxidative stress and that most pesticides, and especially paraquat and cypermethrin, were genotoxic in HepG2 cells. We could also show that mixtures dominated by these two pesticides displayed a marked genotoxic potency, which agreed with our previous population studies.

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Ultraviolet (UV)B radiation affects plant growth inhibiting cell proliferation. This inhibition is in part controlled by the activity of transcription factors from the E2F family. In particular, the participation of E2Fc and E2Fe in UV-B responses in Arabidopsis plants was previously reported. However, the E2Fa and E2Fb contribution to these processes has still not been investigated. Thus, in this work, we provide evidence that, in Arabidopsis, both E2Fa and E2Fb control leaf size under UV-B conditions without participating in the repair of cyclobutane pyrimidine dimers in the DNA. Nevertheless, in UV-B-exposed seedlings, E2Fa, but not E2Fb, regulates primary root elongation, cell proliferation, and programmed cell death in the meristematic zone. Using e2fa mutants that overexpress E2Fb, we showed that the role of E2Fa in the roots could not be replaced by E2Fb. Finally, our results show that E2Fa and E2Fb differentially regulate the expression of genes that activate the DNA damage response and cell cycle progression, both under conditions without UV-B and after exposure. Overall, we showed that both E2Fa and E2Fb have different and non-redundant roles in developmental and DNA damage responses in Arabidopsis plants exposed to UV-B.

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Cellular function is highly dependent on genomic stability, which is mainly ensured by two cellular mechanisms: the DNA damage response (DDR) and the Spindle Assembly Checkpoint (SAC). The former provides the repair of damaged DNA, and the latter ensures correct chromosome segregation. This review focuses on recently emerging data indicating that the SAC and the DDR proteins function together throughout the cell cycle, suggesting crosstalk between both checkpoints to maintain genome stability.

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Radiation therapy has been used worldwide for many decades as a therapeutic regimen for the treatment of different types of cancer. Just over 50% of cancer patients are treated with radiotherapy alone or with other types of antitumor therapy. Radiation can induce different types of cell damage: directly, it can induce DNA single- and double-strand breaks; indirectly, it can induce the formation of free radicals, which can interact with different components of cells, including the genome, promoting structural alterations. During treatment, radiosensitive tumor cells decrease their rate of cell proliferation through cell cycle arrest stimulated by DNA damage. Then, DNA repair mechanisms are turned on to alleviate the damage, but cell death mechanisms are activated if damage persists and cannot be repaired. Interestingly, some cells can evade apoptosis because genome damage triggers the cellular overactivation of some DNA repair pathways. Additionally, some surviving cells exposed to radiation may have alterations in the expression of tumor suppressor genes and oncogenes, enhancing different hallmarks of cancer, such as migration, invasion, and metastasis. The activation of these genetic pathways and other epigenetic and structural cellular changes in the irradiated cells and extracellular factors, such as the tumor microenvironment, is crucial in developing tumor radioresistance. The tumor microenvironment is largely responsible for the poor efficacy of antitumor therapy, tumor relapse, and poor prognosis observed in some patients. In this review, we describe strategies that tumor cells use to respond to radiation stress, adapt, and proliferate after radiotherapy, promoting the appearance of tumor radioresistance. Also, we discuss the clinical impact of radioresistance in patient outcomes. Knowledge of such cellular strategies could help the development of new clinical interventions, increasing the radiosensitization of tumor cells, improving the effectiveness of these therapies, and increasing the survival of patients.