RESUMEN
Human genetic syndromes deficient in nucleotide excision repair (NER), such as xeroderma pigmentosum and Cockayne syndrome, may present neurological abnormalities and premature aging symptoms. Unrepaired endogenously generated DNA damage that hampers transcription is a strong candidate that contributes to the development of these severe effects in neuronal tissue. Endogenous lesions include those generated due to byproducts of cellular metabolisms, such as reactive oxygen species. This review presents much of the evidence on the mechanisms related to neurodegenerative processes associated with DNA damage responses. The primary focus is on the effects of the transcription machinery, including the accumulation of DNAâ¢RNA hybrids (R-loops) that, in turn, influence DNA damage and repair metabolism. Moreover, several neuronal tissues present higher expression of long genes, a genomic subset more affected by DNA lesions, which may explain part of the neurological abnormalities in these patients. Also, neuronal tissues have different DNA repair capabilities that might result in different neurological consequences, as observed in patients and NER deficient animal models. The better understanding of how the accumulation of transcription blocking lesions can lead to neurological abnormalities and premature aging-like phenotypes may assist us in finding potential biomarkers and therapeutic targets that might improve the lives of these patients, as well as other neurological disorders in the general population.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/genética , Enfermedades del Sistema Nervioso/genética , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the 'A-rule'. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.
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Mutagénesis/genética , Estrés Oxidativo/genética , Dímeros de Pirimidina/genética , Xerodermia Pigmentosa/genética , Línea Celular , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , Humanos , Mutagénesis/efectos de la radiación , Mutación/genética , Mutación/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Dímeros de Pirimidina/efectos de la radiación , Rayos Ultravioleta , Secuenciación del Exoma , Xerodermia Pigmentosa/etiologíaRESUMEN
Skin melanocytes harbor a complex photosensitive system comprised of opsins, which were shown, in recent years, to display light- and thermo-independent functions. Based on this premise, we investigated whether melanopsin, OPN4, displays such a role in normal melanocytes. In this study, we found that murine Opn4KO melanocytes displayed a faster proliferation rate compared to Opn4WT melanocytes. Cell cycle population analysis demonstrated that OPN4KO melanocytes exhibited a faster cell cycle progression with reduced G0-G1, and highly increased S and slightly increased G2/M cell populations compared to the Opn4WT counterparts. Expression of specific cell cycle-related genes in Opn4KO melanocytes exhibited alterations that corroborate a faster cell cycle progression. We also found significant modification in gene and protein expression levels of important regulators of melanocyte physiology. PER1 protein level was higher while BMAL1 and REV-ERBα decreased in Opn4KO melanocytes compared to Opn4WT cells. Interestingly, the gene expression of microphthalmia-associated transcription factor (MITF) was upregulated in Opn4KO melanocytes, which is in line with a higher proliferative capability. Taken altogether, we demonstrated that OPN4 regulates cell proliferation, cell cycle, and affects the expression of several important factors of the melanocyte physiology; thus, arguing for a putative tumor suppression role in melanocytes.
Asunto(s)
Ciclo Celular/genética , Melanocitos/metabolismo , Opsinas de Bastones/deficiencia , Animales , Biomarcadores , Proteínas CLOCK/genética , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/genética , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Melanocitos/efectos de los fármacos , Ratones , Piel/citología , Piel/metabolismoRESUMEN
PURPOSE: To identify novel genes associated with intellectual disability (ID) in four unrelated families. METHODS: Here, through exome sequencing and international collaboration, we report eight individuals from four unrelated families of diverse geographic origin with biallelic loss-of-function variants in UBE4A. RESULTS: Eight evaluated individuals presented with syndromic intellectual disability and global developmental delay. Other clinical features included hypotonia, short stature, seizures, and behavior disorder. Characteristic features were appreciated in some individuals but not all; in some cases, features became more apparent with age. We demonstrated that UBE4A loss-of-function variants reduced RNA expression and protein levels in clinical samples. Mice generated to mimic patient-specific Ube4a loss-of-function variant exhibited muscular and neurological/behavioral abnormalities, some of which are suggestive of the clinical abnormalities seen in the affected individuals. CONCLUSION: These data indicate that biallelic loss-of-function variants in UBE4A cause a novel intellectual disability syndrome, suggesting that UBE4A enzyme activity is required for normal development and neurological function.
Asunto(s)
Enanismo , Discapacidad Intelectual , Ubiquitina-Proteína Ligasas/genética , Animales , Niño , Discapacidades del Desarrollo/genética , Humanos , Discapacidad Intelectual/genética , Ratones , Hipotonía Muscular , Fenotipo , Síndrome , Secuenciación del ExomaRESUMEN
BACKGROUND: Members of the family of NEK protein kinases (NIMA-related kinases) were described to have crucial roles in regulating different aspects of the cell cycle. NEK10 was reported to take part in the maintenance of the G2/M checkpoint after exposure to ultraviolet light. NEK1, NEK5, NEK2 and NEK4 proteins on the other hand have been linked to mitochondrial functions. METHODS: HEK293T cells were transfected with FLAG empty vector or FLAG-NEK10 and treated or not with Zeocin. For proteomic analysis, proteins co-precipitated with the FLAG constructs were digested by trypsin, and then analyzed via LC-MS/MS. Proteomic data retrieved were next submitted to Integrated Interactome System analysis and differentially expressed proteins were attributed to Gene Ontology biological processes and assembled in protein networks by Cytoscape. For functional, cellular and molecular analyses two stable Nek10 silenced HeLa cell clones were established. RESULTS: Here, we discovered the following possible new NEK10 protein interactors, related to mitochondrial functions: SIRT3, ATAD3A, ATAD3B, and OAT. After zeocin treatment, the spectrum of mitochondrial interactors increased by the proteins: FKBP4, TXN, PFDN2, ATAD3B, MRPL12, ATP5J, DUT, YWHAE, CS, SIRT3, HSPA9, PDHB, GLUD1, DDX3X, and APEX1. We confirmed the interaction of NEK10 and GLUD1 by proximity ligation assay and confocal microscopy. Furthermore, we demonstrated that NEK10-depleted cells showed more fragmented mitochondria compared to the control cells. The knock down of NEK10 resulted further in changes in mitochondrial reactive oxygen species (ROS) levels, decreased citrate synthase activity, and culminated in inhibition of mitochondrial respiration, affecting particularly ATP-linked oxygen consumption rate and spare capacity. NEK10 depletion also decreased the ratio of mtDNA amplification, possibly due to DNA damage. However, the total mtDNA content increased, suggesting that NEK10 may be involved in the control of mtDNA content. CONCLUSIONS: Taken together these data place NEK10 as a novel regulatory player in mitochondrial homeostasis and energy metabolism.
RESUMEN
Cells are daily submitted to high levels of DNA lesions that trigger complex pathways and cellular responses by cell cycle arrest, apoptosis, alterations in transcriptional response, and the onset of DNA repair. Members of the NIMA-related kinase (NEK) family have been related to DNA damage response and repair and the first insight about NEK5 in this context is related to its role in centrosome separation resulting in defects in chromosome integrity. Here we investigate the potential correlation between NEK5 and the DNA damage repair index. The effect of NEK5 in double-strand breaks caused by etoposide was accessed by alkaline comet assay and revealed that NEK5-silenced cells are more sensitive to etoposide treatment. Topoisomerase IIß (TOPIIß) is a target of etoposide that leads to the production of DNA breaks. We demonstrate that NEK5 interacts with TOPIIß, and the dynamics of this interaction is evaluated by proximity ligation assay. The complex NEK5/TOPIIß is formed immediately after etoposide treatment. Taken together, the results of our study reveal that NEK5 depletion increases DNA damage and impairs proper DNA damage response, pointing out NEK5 as a potential kinase contributor to genomic stability.
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Daño del ADN/genética , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Etopósido/farmacología , Quinasas Relacionadas con NIMA/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , ADN/efectos de los fármacos , ADN/genética , Células HEK293 , Humanos , Quinasas Relacionadas con NIMA/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Horizontal gene transfer (HGT) has a major impact on the evolution of prokaryotic genomes, as it allows genes evolved in different contexts to be combined in a single genome, greatly enhancing the ways evolving organisms can explore the gene content space and adapt to the environment. A systematic analysis of HGT in a large number of genomes is of key importance in understanding the impact of HGT in the evolution of prokaryotes. We developed a method for the detection of genes that potentially originated by HGT based on the comparison of BLAST scores between homologous genes to 16S rRNA-based phylogenetic distances between the involved organisms. The approach was applied to 697 prokaryote genomes and estimated that in average approximately 15% of the genes in prokaryote genomes originated by HGT, with a clear correlation between the proportion of predicted HGT genes and the size of the genome. The methodology was strongly supported by evolutionary relationships, as tested by the direct phylogenetic reconstruction of many of the HGT candidates. Studies performed with Escherichia coli W3110 genome clearly show that HGT proteins have fewer interactions when compared to those predicted as vertical inherited, an indication that the number of protein partners imposes limitations to horizontal transfer. A detailed functional classification confirms that genes related to protein translation are vertically inherited, whereas interestingly, transport and binding proteins are strongly enriched among HGT genes. Because these genes are related to the cell exchange with their environment, their transfer most likely contributed to successful adaptation throughout evolution.
Asunto(s)
Evolución Molecular , Transferencia de Gen Horizontal , Genoma Bacteriano , Células Procariotas , Bacterias/genética , Escherichia coli/genética , Filogenia , ARN Ribosómico 16S/genéticaRESUMEN
Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Proteínas Nucleares/genética , Nucleotidiltransferasas/genética , Adenoviridae/genética , Adenoviridae/metabolismo , Línea Celular Transformada , Daño del ADN , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribodipirimidina Fotoliasa , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Genoma Humano , Humanos , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Dímeros de Pirimidina/metabolismo , Fase S/genética , Transducción Genética , Rayos UltravioletaRESUMEN
Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here, we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However, iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover, these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells, regulating the expression of TP53 and TXNIP and ROS production.
Asunto(s)
Envejecimiento Prematuro/patología , Síndrome de Cockayne/patología , Células Madre Pluripotentes Inducidas/patología , Estrés Oxidativo , Muerte Celular/genética , Hipoxia de la Célula/genética , Supervivencia Celular/genética , Células Clonales , Síndrome de Cockayne/genética , Daño del ADN/genética , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The ultraviolet (UV) component of sunlight can damage DNA. Although most solar UV is absorbed by the ozone layer, wavelengths > 300 nm (UVA and UVB bands) can reach the Earth's surface. It is essential to understand the genotoxic effects of UV light, particularly in natural environments. Caulobacter crescentus, a bacterium widely employed as a model for cell cycle studies, was selected for this study. Strains proficient and deficient in DNA repair (uvrA-) were used to concurrently investigate three genotoxic endpoints: cytotoxicity, SOS induction, and gene mutation, using colony-formation, the SOS chromotest, and RifR mutagenesis, respectively. Our findings underscore the distinct impacts of individual UV bands and the full spectrum of sunlight itself in C. crescentus. UVC light was highly genotoxic, especially for the repair-deficient strain. A UVB dose equivalent to 20 min sunlight exposure also affected the cells. UVA exposure caused a significant response only at high doses, likely due to activation of photorepair. Exposure to solar irradiation resulted in reduced levels of SOS induction, possibly due to decreased cell survival. However, mutagenicity is increased, particularly in uvrA- deficient cells.
Asunto(s)
Caulobacter crescentus , Rayos Ultravioleta , Rayos Ultravioleta/efectos adversos , Caulobacter crescentus/genética , Daño del ADN , Reparación del ADN , MutaciónRESUMEN
Xeroderma pigmentosum (XP) variant cells are deficient in the translesion synthesis (TLS) DNA polymerase Polη (eta). This protein contributes to DNA damage tolerance, bypassing unrepaired UV photoproducts and allowing S-phase progression with minimal delay. In the absence of Polη, backup polymerases perform TLS of UV lesions. However, which polymerase plays this role in human cells remains an open question. Here, we investigated the potential role of Polι (iota) in bypassing ultraviolet (UV) induced photoproducts in the absence of Polη, using NER-deficient (XP-C) cells knocked down for Polι and/or Polη genes. Our results indicate that cells lacking either Polι or Polη have increased sensitivity to UVC radiation. The lack of both TLS polymerases led to increased cell death and defects in proliferation and migration. Loss of both polymerases induces a significant replication fork arrest and G1/S-phase blockage, compared to the lack of Polη alone. In conclusion, we propose that Polι acts as a bona fide backup for Polη in the TLS of UV-photoproducts.
Asunto(s)
ADN Polimerasa iota , Xerodermia Pigmentosa , Humanos , Daño del ADN , Síntesis Translesional de ADN , Replicación del ADN , Xerodermia Pigmentosa/genética , Rayos Ultravioleta , Reparación del ADNRESUMEN
Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Polκ) has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Polκ in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Polκ knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 µM); (ii) demonstrates significant cytotoxicity (25-200 µM); (iii) exerts antiproliferative effects (≤25 µM) and promotes cell cycle arrest (G2/M phase) in Polκ KO spheroids when compared with WTE counterparts. Furthermore, Polκ KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Polκ KO than in WTE spheroids. Collectively, the results suggest that TLS Polκ plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Polκ emerges as a potential therapeutic target for GBM patients.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Resistencia a Antineoplásicos , Glioblastoma , Esferoides Celulares , Temozolomida , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/enzimología , Temozolomida/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/enzimología , Antineoplásicos Alquilantes/farmacologíaRESUMEN
Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.
Asunto(s)
Metilasas de Modificación del ADN , ADN Polimerasa iota , Enzimas Reparadoras del ADN , ADN Polimerasa Dirigida por ADN , Resistencia a Antineoplásicos , Glioblastoma , Temozolomida , Temozolomida/farmacología , Humanos , Glioblastoma/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Línea Celular Tumoral , Metilasas de Modificación del ADN/metabolismo , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/deficiencia , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Daño del ADN , Supervivencia Celular/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Reparación del ADN , Técnicas de Inactivación de GenesRESUMEN
Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (ðððð¥ ) of 2.0 g L-1 was reached after 5 h of cultivation, corresponding to ðX = 0.4 g L-1 h. The µððð¥ corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.
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Desoxirribodipirimidina Fotoliasa , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Thermus thermophilus , Rayos Ultravioleta , ADN/química , Reparación del ADNRESUMEN
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.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Daño del ADN , Reparación del ADN , Estructuras R-Loop , Transcripción Genética , Trypanosoma cruzi , Rayos Ultravioleta , Trypanosoma cruzi/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas Protozoarias/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Muerte Celular , Apoptosis , HumanosRESUMEN
Solar ultraviolet (UV) radiation is widely known as an environmental genotoxic agent that affects ecosystems and the human population, generating concerns and motivating worldwide scientific efforts to better understand the role of sunlight in the induction of DNA damage, cell death, mutagenesis, and ultimately, carcinogenesis. In this review, general aspects of UV radiation at the Earth's surface are reported, considering measurements by physical and biological sensors that monitor solar UV radiation under different environmental conditions. The formation of DNA photoproducts and other types of DNA damage by different UV wavelengths are compared with the present information on their roles in inducing biological effects. Moreover, the use of DNA-based biological dosimeters is presented as a feasible molecular and cellular tool that is focused on the evaluation of DNA lesions induced by natural sunlight. Clearly, direct environmental measurements demonstrate the biological impact of sunlight in different locations worldwide and reveal how this affects the DNA damage profile at different latitudes. These tools are also valuable for the quantification of photoprotection provided by commercial sunscreens against the induction of DNA damage and cell death, employing DNA repair-deficient cells that are hypersensitive to sunlight. Collectively, the data demonstrate the applicability of DNA-based biosensors as alternative, complementary, and reliable methods for registering variations in the genotoxic impact of solar UV radiation and for determining the level of photoprotection sunscreens provided at the level of DNA damage and cell death.
Asunto(s)
Técnicas Biosensibles/métodos , Daño del ADN/efectos de la radiación , ADN/química , ADN/genética , Radiometría/métodos , Rayos Ultravioleta , Animales , Técnicas Biosensibles/instrumentación , Humanos , Radiometría/instrumentación , Protectores Solares/farmacologíaRESUMEN
UVA light (320-400 nm) represents approximately 95% of the total solar UV radiation that reaches the Earth's surface. UVA light induces oxidative stress and the formation of DNA photoproducts in skin cells. These photoproducts such as pyrimidine dimers (cyclobutane pyrimidine dimers, CPDs, and pyrimidine (6-4) pyrimidone photoproducts, 6-4PPs) are removed by nucleotide excision repair (NER). In this repair pathway, the XPA protein is recruited to the damage removal site; therefore, cells deficient in this protein are unable to repair the photoproducts. The aim of this study was to investigate the involvement of oxidative stress and the formation of DNA photoproducts in UVA-induced cell death. In fact, similar levels of oxidative stress and oxidised bases were detected in XP-A and NER-proficient cells exposed to UVA light. Interestingly, CPDs were detected in both cell lines; however, 6-4PPs were detected only in DNA repair-deficient cells. XP-A cells were also observed to be significantly more sensitive to UVA light compared to NER-proficient cells, with an increased induction of apoptosis, while necrosis was similarly observed in both cell lines. The induction of apoptosis and necrosis in XP-A cells using adenovirus-mediated transduction of specific photolyases was investigated and we confirm that both types of photoproducts are the primary lesions responsible for inducing cell death in XP-A cells and may trigger the skin-damaging effects of UVA light, particularly skin ageing and carcinogenesis.
Asunto(s)
Muerte Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , ADN/química , Estrés Oxidativo/efectos de la radiación , Línea Celular , ADN/genética , Reparación del ADN/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/genética , Expresión Génica , Humanos , Carbonilación Proteica/efectos de la radiación , Rayos UltravioletaRESUMEN
All living cells utilize intricate DNA repair mechanisms to address numerous types of DNA lesions and to preserve genomic integrity, and pluripotent stem cells have specific needs due to their remarkable ability of self-renewal and differentiation into different functional cell types. Not surprisingly, human stem cells possess a highly efficient DNA repair network that becomes less efficient upon differentiation. Moreover, these cells also have an anaerobic metabolism, which reduces the mitochondria number and the likelihood of oxidative stress, which is highly related to genomic instability. If DNA lesions are not repaired, human stem cells easily undergo senescence, cell death or differentiation, as part of their DNA damage response, avoiding the propagation of stem cells carrying mutations and genomic alterations. Interestingly, cancer stem cells and typical stem cells share not only the differentiation potential but also their capacity to respond to DNA damage, with important implications for cancer therapy using genotoxic agents. On the other hand, the preservation of the adult stem cell pool, and the ability of cells to deal with DNA damage, is essential for normal development, reducing processes of neurodegeneration and premature aging, as one can observe on clinical phenotypes of many human genetic diseases with defects in DNA repair processes. Finally, several recent findings suggest that DNA repair also plays a fundamental role in maintaining the pluripotency and differentiation potential of embryonic stem cells, as well as that of induced pluripotent stem (iPS) cells. DNA repair processes also seem to be necessary for the reprogramming of human cells when iPS cells are produced. Thus, the understanding of how cultured pluripotent stem cells ensure the genetic stability are highly relevant for their safe therapeutic application, at the same time that cellular therapy is a hope for DNA repair deficient patients.
Asunto(s)
Diferenciación Celular/genética , Reparación del ADN , Células Madre Pluripotentes/fisiología , Células Madre Adultas , Envejecimiento , Células Madre Embrionarias/fisiología , Inestabilidad Genómica , Humanos , Células Madre Neoplásicas , Estrés Oxidativo/genéticaRESUMEN
CONTEXT: One approach to protect human skin against the dangerous effects of solar ultraviolet (UV) irradiation is the use of natural products, such as photoprotectors. Phyllanthus orbicularis Kunth (Euphorbiaceae) is a Cuban endemic plant used in popular medicine. Its antigenotoxicity effect against some harmful agents has been investigated. However, the effect in ultraviolet B (UVB)-irradiated human cells has not been previously assessed. OBJECTIVE: The protective effect of a P. orbicularis extract against UVB light-induced damage in human cells was evaluated. MATERIALS AND METHODS: DNA repair proficient (MRC5-SV) and deficient (XP4PA, complementation group XPC) cell-lines were used. Damaging effects of UVB light were evaluated by clonogenic assay and apoptosis induction by flow cytometry techniques. The extent of DNA repair itself was determined by the removal of cyclobutane pyrimidine dimers (CPDs). The CPDs were detected and quantified by slot-blot assay. RESULTS: Treatment of UVB-irradiated MRC5-SV cells with P. orbicularis extract increased the percentage of colony-forming cells from 36.03 ± 3.59 and 4.42 ± 1.45 to 53.14 ± 8.8 and 14.52 ± 1.97, for 400 and 600 J/m(2), respectively. A decrease in apoptotic cell population was observed in cells maintained within the extract. The P. orbicularis extract enhanced the removal of CPD from genomic DNA. The CPDs remaining were found to be about 27.7 and 1.1%, while with plant extract, treatment these values decreased to 16.1 and 0.2%, for 3 and 24 h, respectively. DISCUSSION AND CONCLUSION: P. orbicularis aqueous extract protects human cells against UVB damage. This protective effect is through the modulation of DNA repair effectiveness.
Asunto(s)
Reparación del ADN/efectos de los fármacos , Phyllanthus/química , Extractos Vegetales/farmacología , Rayos Ultravioleta/efectos adversos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Cuba , Reparación del ADN/efectos de la radiación , Citometría de Flujo , Humanos , Dímeros de Pirimidina/metabolismo , Factores de TiempoRESUMEN
DNA-targeting agents have a significant clinical use, although toxicity remains an issue that plays against their widespread application. Understanding the mechanism of action and DNA damage response elicited by such compounds might contribute to the improvement of their use in anticancer chemotherapy. In a previous study, our research group characterized a new DNA-targeting agent - pradimicin-IRD. Since DNA-targeting agents and DNA repair are close-related subjects, the present study used in silico-modelling and a transcriptomic approach seeking to characterize the DNA repair pathways activated in HCT 116 cells following pradimicin-IRD treatment. Molecular docking analysis showed pradimicin-IRD as a DNA intercalating agent and a potential inhibitor of DNA-binding proteins. Furthermore, the transcriptomic study highlighted DNA repair functions related to genes modulated by pradimicin-IRD, such as nucleotide excision repair, telomeres maintenance and double-strand break repair. When validating these functions, PCNA protein levels decreased after exposure to pradimicin. Furthermore, molecular docking analysis suggested DNA-pradimicin-PCNA interaction. In addition, hTERT and POLH showed reduced mRNA levels after 6 h of treatment with pradimicin-IRD. Moreover, POLH-deficient cells displayed higher resistance to pradimicin-IRD than POLH-proficient cells and the compound prevented formation of the POLH/DNA complex (molecular docking). Since the modulation of DNA repair genes by pradimicin-IRD is TP53-independent, unlike doxorubicin, dissimilarities between the mechanism of action and the DNA damage response of pradimicin-IRD and doxorubicin open new insights for further studies of pradimicin-IRD as a new antineoplastic compound.