RESUMEN
Human genome stability requires efficient repair of oxidized bases, which is initiated via damage recognition and excision by NEIL1 and other base excision repair (BER) pathway DNA glycosylases (DGs). However, the biological mechanisms underlying detection of damaged bases among the million-fold excess of undamaged bases remain enigmatic. Indeed, mutation rates vary greatly within individual genomes, and lesion recognition by purified DGs in the chromatin context is inefficient. Employing super-resolution microscopy and co-immunoprecipitation assays, we find that acetylated NEIL1 (AcNEIL1), but not its non-acetylated form, is predominantly localized in the nucleus in association with epigenetic marks of uncondensed chromatin. Furthermore, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) revealed non-random AcNEIL1 binding near transcription start sites of weakly transcribed genes and along highly transcribed chromatin domains. Bioinformatic analyses revealed a striking correspondence between AcNEIL1 occupancy along the genome and mutation rates, with AcNEIL1-occupied sites exhibiting fewer mutations compared to AcNEIL1-free domains, both in cancer genomes and in population variation. Intriguingly, from the evolutionarily conserved unstructured domain that targets NEIL1 to open chromatin, its damage surveillance of highly oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated â¼500 million years ago during the buildup of free atmospheric oxygen.
Asunto(s)
Cromatina/fisiología , ADN Glicosilasas/metabolismo , Reparación del ADN , Procesamiento Proteico-Postraduccional , Acetilación , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/ultraestructura , ADN Glicosilasas/química , ADN Glicosilasas/fisiología , Reparación del ADN/genética , Conjuntos de Datos como Asunto , Evolución Molecular , Genes de Helminto , Genes Homeobox , Células HEK293 , Proteínas del Helminto/genética , Humanos , Invertebrados/genética , Invertebrados/metabolismo , Lisina/química , Mutación , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidad , Oxidación-Reducción , Proteoma , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sitio de Iniciación de la Transcripción , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
The most frequent DNA lesion resulting from an oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoG). 8-oxoG is a premutagenic base modification due to its capacity to pair with adenine. Thus, the repair of 8-oxoG is critical for the preservation of the genetic information. Nowadays, 8-oxoG is also considered as an oxidative stress-sensor with a putative role in transcription regulation. In mammalian cells, the modified base is excised by the 8-oxoguanine DNA glycosylase (OGG1), initiating the base excision repair (BER) pathway. OGG1 confronts the massive challenge that is finding rare occurrences of 8-oxoG among a million-fold excess of normal guanines. Here, we review the current knowledge on the search and discrimination mechanisms employed by OGG1 to find its substrate in the genome. While there is considerable data from in vitro experiments, much less is known on how OGG1 is recruited to chromatin and scans the genome within the cellular nucleus. Based on what is known of the strategies used by proteins searching for rare genomic targets, we discuss the possible scenarios allowing the efficient detection of 8-oxoG by OGG1.
Asunto(s)
ADN Glicosilasas/metabolismo , Guanina/análogos & derivados , Animales , ADN/metabolismo , ADN Glicosilasas/genética , ADN Glicosilasas/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Guanina/metabolismo , Guanina/fisiología , Humanos , Estrés Oxidativo/fisiologíaRESUMEN
Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.
Asunto(s)
Daño del ADN , ADN Glicosilasas/metabolismo , Reparación del ADN , Microscopía de Fuerza Atómica , Conformación de Ácido Nucleico , Automatización/métodos , ADN Glicosilasas/fisiología , Reparación del ADN/fisiología , Ensayos Analíticos de Alto Rendimiento , Humanos , Microscopía de Fuerza Atómica/métodosRESUMEN
8-oxoguanine (8-oxoG) is one of the most prevalent genotoxic lesions, and it is generated in DNA attacked by reactive oxygen species (ROS). Adenine misincorporated opposite to 8-oxoG during replication is excised by MutY homolog (MUTYH), an important protein of the base excision repair (BER) system. Mutyh plays an important role in the maintenance of genomic integrity, but the functional consequences of Mutyh deficiency are not fully understood. In the current study, we investigated the histological and functional changes of five tissues (hippocampus, heart, liver, kidney and lung) and their molecular basis in Mutyh-/- and wild-type mice exposed to D-galactose (D-gal). Our data indicated that Mutyh deficiency hindered the weight gain of experimental mice and induced substantial alterations of 8-oxoG content and superoxide dismutase (SOD) activity, but no significant histological and functional impairment appeared in the investigated tissues of Mutyh- deficient mice without D-gal exposure. Under low-dose D-gal exposure, Mutyh deficiency altered expression of genes involved in mitochondrial unfolded protein response (UPRmt) in the heart, liver and lung, and caused an enhanced expression of mitochondrial dynamics proteins (MDPs) in hippocampus and liver. The stress responses could maintain mitochondrial proteostasis and function. However, such responses were not noted when experiencing excessive damage burden induced by high-dose D-gal exposure, in which Mutyh deficiency increased accumulation of 8-oxoG and aggravated mitonuclear protein imbalance, as well as histological lesions in heart, liver and kidney. A higher sensitivity to ROS-induced cardiotoxicity with high-dose D-gal exposure was noticed in Mutyh-/- mice. However, no differences in learning and memory impairments were observed between Mutyh-/- and wild-type mice with high-dose D-gal exposure. In conclusion, our data demonstrated that Mutyh deficiency has different impacts on various tissues based on the degree of oxidative stress.
Asunto(s)
Conducta Animal/efectos de los fármacos , Daño del ADN , ADN Glicosilasas/fisiología , Mitocondrias/patología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Disfunción Ventricular Izquierda/etiología , Animales , Galactosa/farmacología , Guanina/análogos & derivados , Guanina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Superóxido Dismutasa/metabolismo , Disfunción Ventricular Izquierda/patologíaRESUMEN
The common oxidatively generated lesion, 8-oxo-7,8-dihydroguanine (8-oxoGua), is removed from DNA by base excision repair. The glycosylase primarily charged with recognition and removal of this lesion is 8-oxoGuaDNA glycosylase 1 (OGG1). When left unrepaired, 8-oxodG alters transcription and is mutagenic. Individuals homozygous for the less active OGG1 allele, Ser326Cys, have increased risk of several cancers. Here, small molecule enhancers of OGG1 were identified and tested for their ability to stimulate DNA repair and protect cells from the environmental hazard paraquat (PQ). PQ-induced mtDNA damage was inversely proportional to the levels of OGG1 expression whereas stimulation of OGG1, in some cases, entirely abolished its cellular effects. The PQ-mediated decline of mitochondrial membrane potential or nuclear condensation were prevented by the OGG1 activators. In addition, in Ogg1-/- mouse embryonic fibroblasts complemented with hOGG1S326C, there was increased cellular and mitochondrial reactive oxygen species compared to their wild type counterparts. Mitochondrial extracts from cells expressing hOGG1S326C were deficient in mitochondrial 8-oxodG incision activity, which was rescued by the OGG1 activators. These data demonstrate that small molecules can stimulate OGG1 activity with consequent cellular protection. Thus, OGG1-activating compounds may be useful in select humans to mitigate the deleterious effects of environmental oxidants and mutagens.
Asunto(s)
ADN Glicosilasas/metabolismo , ADN Glicosilasas/fisiología , Reparación del ADN , ADN Mitocondrial/genética , Mitocondrias/efectos de los fármacos , Serina/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Células A549 , Animales , Células Cultivadas , Daño del ADN , ADN Glicosilasas/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Herbicidas/efectos adversos , Humanos , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Mutación , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Paraquat/efectos adversos , Especies Reactivas de Oxígeno/metabolismo , Serina/genéticaRESUMEN
PURPOSE: To determine whether low dose/low dose rate radiation-induced genetic instability may result from radiation-induced inactivation of mechanisms induced by the ATM-dependent DNA damage response checkpoint. To this end, we analysed the faithfulness of T cell receptor (TR) gene rearrangement by V(D)J recombination in DNA from mice exposed to a single dose of X-ray or chronically exposed to low dose rate γ radiation. MATERIALS AND METHODS: Genomic DNA obtained from the blood or the thymus of wild type or Ogg1-deficient mice exposed to low (0.1) or intermediate/high (0.2-1 Gy) doses of radiation either by acute X-rays exposure or protracted exposure to low dose-rate γ-radiation was used to analyse by PCR the presence of illegitimate TR gene rearrangements. RESULTS: Radiation exposure does not increase the onset of TR gene trans-rearrangements in irradiated mice. In mice where it happens, trans-rearrangements remain sporadic events in developing T lymphocytes. CONCLUSION: We concluded that low dose/low dose rate ionizing radiation (IR) exposure does not lead to widespread inactivation of ATM-dependent mechanisms, and therefore that the mechanisms enforcing genetic stability are not impaired by IR in developing lymphocytes and lymphocyte progenitors, including BM-derived hematopoietic stem cells, in low dose/low dose rate exposed mice.
Asunto(s)
Reordenamiento Génico , Genes Codificadores de los Receptores de Linfocitos T/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , ADN Glicosilasas/fisiología , Inestabilidad Genómica , Linfocitos/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos CBA , Radiación Ionizante , Rayos XRESUMEN
OGG1 (8-oxoguanine-DNA glycosylase) is the major DNA repair glycosylase removing the premutagenic DNA base modification 8-oxo-7,8-dihydroguanine (8-oxoG) from the genome of mammalian cells. In addition, there is accumulating evidence that OGG1 and its substrate 8-oxoG might function in the regulation of certain genes, which could account for an attenuated immune response observed in Ogg1-/- mice in several settings. Indications for at least two different mechanisms have been obtained. Thus, OGG1 could either act as an ancillary transcription factor cooperating with the lysine-specific demethylase LSD1 or as an activator of small GTPases. Here, we analysed the activation by lipopolysaccaride (LPS) of primary splenocytes obtained from two different Ogg1-/- mouse strains. We found that the induction of TNF-α expression was reduced in splenocytes (in particular macrophages) of both Ogg1-/- strains. Notably, an inhibitor of LSD1, OG-L002, reduced the induction of TNF-α mRNA in splenocytes from wild-type mice to the level observed in splenocytes from Ogg1-/- mice and had no influence in the latter cells. In contrast, inhibitors of the MAP kinases p38 and JNK as well as the antioxidant N-acetylcysteine attenuated the LPS-stimulated TNF-α expression both in the absence and presence of OGG1. The free base 8-oxo-7,8-dihydroguanine had no influence on the TNF-α expression in the splenocytes. The data demonstrate that OGG1 plays a role in an LSD1-dependent pathway of LPS-induced macrophage activation in mice.
Asunto(s)
ADN Glicosilasas/inmunología , Bazo/inmunología , Factor de Necrosis Tumoral alfa/genética , Animales , ADN/metabolismo , Daño del ADN , ADN Glicosilasas/metabolismo , ADN Glicosilasas/fisiología , Reparación del ADN , Regulación de la Expresión Génica , Guanina/análogos & derivados , Guanina/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Bazo/citología , Bazo/metabolismo , Factores de Transcripción/inmunología , Factores de Transcripción/fisiologíaRESUMEN
Global distribution of hepatocellular carcinomas (HCCs) is dominated by its incidence in developing countries, accounting for >700,000 estimated deaths per year, with dietary exposures to aflatoxin (AFB1) and subsequent DNA adduct formation being a significant driver. Genetic variants that increase individual susceptibility to AFB1-induced HCCs are poorly understood. Herein, it is shown that the DNA base excision repair (BER) enzyme, DNA glycosylase NEIL1, efficiently recognizes and excises the highly mutagenic imidazole ring-opened AFB1-deoxyguanosine adduct (AFB1-Fapy-dG). Consistent with this in vitro result, newborn mice injected with AFB1 show significant increases in the levels of AFB1-Fapy-dG in Neil1-/- vs. wild-type liver DNA. Further, Neil1-/- mice are highly susceptible to AFB1-induced HCCs relative to WT controls, with both the frequency and average size of hepatocellular carcinomas being elevated in Neil1-/- The magnitude of this effect in Neil1-/- mice is greater than that previously measured in Xeroderma pigmentosum complementation group A (XPA) mice that are deficient in nucleotide excision repair (NER). Given that several human polymorphic variants of NEIL1 are catalytically inactive for their DNA glycosylase activity, these deficiencies may increase susceptibility to AFB1-associated HCCs.
Asunto(s)
Aflatoxinas/toxicidad , Carcinoma Hepatocelular/prevención & control , Aductos de ADN/efectos de los fármacos , ADN Glicosilasas/fisiología , Neoplasias Hepáticas Experimentales/prevención & control , Sustancias Protectoras/farmacología , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Femenino , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Venenos/toxicidadRESUMEN
8-Oxo-2'-deoxyguanosine (8-oxo-dG) is a representative of nucleoside damage, which is generated by the reaction of the 8 position of dG with reactive oxygen species. Abundant 8-oxo-dG in DNA exhibits genotoxicity and has been linked to aging and disease, such as cancer. As the metabolism of cancer cells is much faster than that of normal cells, the oxidized product of the oligonucleotides and the nucleotide pool produces 8-oxo-dG and 8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), respectively. Human oxoguanine glycosylase (hOGG1) shows base excision activity for 8-oxo-dG in duplex DNA. On the other hand, human mutT homologue protein (hMTH1, also known as NUDT1) is important for oxidized nucleotide removal including 8-oxo-dGTP, and it is reported that the presence of hMTH1 is not essential for normal cells but is required for the survival of cancer cells. Therefore, we designed and synthesized 8-halogenated 7-deaza-2'-deoxyguanosine triphosphate (8-halo-7-deaza-dGTP) derivatives as mimics of 8-oxo-dGTP in order to interact with hMTH1. The 8-halo-7-deaza-dGTP derivatives were poor substrates for but strong binders to hMTH1. Interestingly, they exhibited strong competitive inhibition of hMTH1 in the hydrolysis of 8-oxo-dGTP. This inhibitory effect is caused by the slower rate of hydrolysis due to possible small enzyme structural changes. Although the detailed inhibition mechanism of the hydrolysis activity of hMTH1 is unknown, this result is the first to demonstrate the potential of nucleoside triphosphate derivatives as antitumor agents.
Asunto(s)
ADN/genética , Desoxiadenosinas , Nucleótidos de Desoxiguanina , Nucleósidos , Tubercidina/análogos & derivados , Antineoplásicos , ADN Glicosilasas/fisiología , Enzimas Reparadoras del ADN/fisiología , Humanos , Hidrólisis , Neoplasias/genética , Neoplasias/metabolismo , Oxidación-Reducción , Monoéster Fosfórico Hidrolasas/fisiología , Especies Reactivas de Oxígeno , Tubercidina/síntesis químicaRESUMEN
Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1(-/-)) and control animals (Ogg1(+/-)). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBC(CD24-)) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.
Asunto(s)
Células Sanguíneas/efectos de la radiación , Daño del ADN/efectos de la radiación , ADN Glicosilasas/fisiología , Reparación del ADN/efectos de la radiación , Rayos gamma/efectos adversos , Animales , ADN Glicosilasas/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Estrés Oxidativo/efectos de la radiación , Especies Reactivas de Oxígeno/metabolismo , Selenio/deficienciaRESUMEN
Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.
Asunto(s)
Reparación del ADN , Ribonucleoproteína Heterogénea-Nuclear Grupo U/fisiología , Autoantígeno Ku/fisiología , Traumatismos por Radiación/etiología , Roturas del ADN de Doble Cadena , ADN Glicosilasas/fisiología , Enzimas Reparadoras del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/fisiología , Proteína Quinasa Activada por ADN/fisiología , Proteínas de Unión al ADN/fisiología , Células HEK293 , Humanos , Fosforilación , Tolerancia a RadiaciónRESUMEN
In plants, genomic DNA methylation which contributes to development and stress responses can be actively removed by DEMETER-like DNA demethylases (DMLs). Indeed, in Arabidopsis DMLs are important for maternal imprinting and endosperm demethylation, but only a few studies demonstrate the developmental roles of active DNA demethylation conclusively in this plant. Here, we show a direct cause and effect relationship between active DNA demethylation mainly mediated by the tomato DML, SlDML2, and fruit ripening- an important developmental process unique to plants. RNAi SlDML2 knockdown results in ripening inhibition via hypermethylation and repression of the expression of genes encoding ripening transcription factors and rate-limiting enzymes of key biochemical processes such as carotenoid synthesis. Our data demonstrate that active DNA demethylation is central to the control of ripening in tomato.
Asunto(s)
ADN Glicosilasas/fisiología , Metilación de ADN , Frutas/fisiología , Proteínas de Plantas/fisiología , Solanum lycopersicum/enzimología , ADN Glicosilasas/genética , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Interferencia de ARNRESUMEN
Ionizing radiations such as X-ray and γ-ray can directly or indirectly produce clustered or multiple damages in DNA. Previous studies have reported that overexpression of DNA glycosylases in Escherichia coli (E. coli) and human lymphoblast cells caused increased sensitivity to γ-ray and X-ray irradiation. However, the effects and the mechanisms of other radiation, such as low dose rate radiation, heavy-ion beams, or hydrogen peroxide (H2O2), are still poorly understood. In the present study, we constructed a stable HeLaS3 cell line overexpressing human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) protein. We determined the survival of HeLaS3 and HeLaS3/hOGG1 cells exposed to UV, heavy-ion beams, γ-rays, and H2O2. The results showed that HeLaS3 cells overexpressing hOGG1 were more sensitive to γ-rays, OH(â¢), and H2O2, but not to UV or heavy-ion beams, than control HeLaS3. We further determined the levels of 8-oxoG foci and of chromosomal double-strand breaks (DSBs) by detecting γ-H2AX foci formation in DNA. The results demonstrated that both γ-rays and H2O2 induced 8-oxoguanine (8-oxoG) foci formation in HeLaS3 cells. hOGG1-overexpressing cells had increased amounts of γ-H2AX foci and decreased amounts of 8-oxoG foci compared with HeLaS3 control cells. These results suggest that excess hOGG1 removes the oxidatively damaged 8-oxoG in DNA more efficiently and therefore generates more DSBs. Micronucleus formation also supported this conclusion. Low dose-rate γ-ray effects were also investigated. We first found that overexpression of hOGG1 also caused increased sensitivity to low dose rate γ-ray irradiation. The rate of micronucleus formation supported the notion that low dose rate irradiation increased genome instability.
Asunto(s)
Reparación del ADN , Rayos gamma/efectos adversos , Iones Pesados/efectos adversos , Peróxido de Hidrógeno/farmacología , Radical Hidroxilo/efectos adversos , Estrés Oxidativo , Tolerancia a Radiación/genética , Rayos Ultravioleta/efectos adversos , Biomarcadores , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Glicosilasas/genética , ADN Glicosilasas/fisiología , Inducción Enzimática , Guanina/análogos & derivados , Guanina/análisis , Células HeLa , Histonas/análisis , Humanos , Pruebas de Micronúcleos , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Oxidative DNA damage accumulation has been implicated in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. The base excision repair pathway is a primary responder to oxidative DNA damage. Effects of loss of base excision repair on normal brain function is a relatively nascent area of research that needs further exploration for better understanding of related brain diseases. Recently, we found that loss of a versatile DNA glycosylase endonuclease 8-like 1 (NEIL1) causes deficits in spatial memory retention using the Morris water maze test. Furthermore, we found that there is a significant loss of NEIL1 enzyme levels and its activity in postmortem Alzheimer's disease brains. Based on the Allen Brain Atlas in situ hybridization data, the expression levels of Neil1 messenger RNA are higher in the olfactory bulb compared with other areas of the brain. Olfaction in mice is a central brain function that involves many central nervous system pathways. Here, we studied the effect of complete loss of Neil1 gene on olfactory function. We explored olfactory function in mice with 3 different behavioral tests namely, olfactory sensitivity, performance, and buried food tests. Neil1(-/-) mice performed poorly compared with wild-type mice in all 3 tests. Our data indicate that loss of Neil1 causes olfactory function deficits supporting our previous findings and that normal brain function requires robust DNA repair.
Asunto(s)
ADN Glicosilasas/fisiología , Trastornos del Olfato/genética , Olfato/genética , Animales , Daño del ADN/genética , Reparación del ADN/genética , Ratones Transgénicos , Estrés Oxidativo/genéticaRESUMEN
Studies in mice with deficient antioxidative enzymes have shown that physiological levels of reactive oxygen species (ROS) can adversely affect the developing embryo and fetus. Herein, DNA repair-deficient progeny of oxoguanine glycosylase 1 (ogg1)-knockout mice lacking repair of the oxidative DNA lesion 8-oxo-2'-deoxyguanosine (8-oxodGuo) exhibited enhanced postnatal neurodevelopmental deficits, revealing the pathogenic potential of 8-oxodGuo initiated by physiological ROS production in fetal brain and providing the first evidence of a pathological phenotype for ogg1-knockout mice. Moreover, when exposed in utero to ethanol (EtOH), ogg1-knockout progeny exhibited higher levels of 8-oxodGuo in fetal brain and more severe postnatal neurodevelopmental deficits than wild-type littermates, both of which were blocked by pretreatment with the free radical trapping agent phenylbutylnitrone. These results suggest that ROS-initiated DNA oxidation, as distinct from altered signal transduction, contributes to neurodevelopmental deficits caused by in utero EtOH exposure, and fetal DNA repair is a determinant of risk.
Asunto(s)
Reacción de Prevención/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Daño del ADN/efectos de los fármacos , ADN Glicosilasas/fisiología , Embrión de Mamíferos/citología , Etanol/toxicidad , Estrés Oxidativo/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Encéfalo/efectos de los fármacos , Depresores del Sistema Nervioso Central/toxicidad , Óxidos N-Cíclicos/farmacología , Reparación del ADN/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Embrión de Mamíferos/efectos de los fármacos , Femenino , Feto/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Fenotipo , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Efectos Tardíos de la Exposición Prenatal/tratamiento farmacológico , Especies Reactivas de OxígenoRESUMEN
Many, if not all, environmental pollutants/chemicals and infectious agents increase intracellular levels of reactive oxygen species (ROS) at the site of exposure. ROS not only function as intracellular signaling entities, but also induce damage to cellular molecules including DNA. Among the several dozen ROS-induced DNA base lesions generated in the genome, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant because of guanine's lowest redox potential among DNA bases. In mammalian cells, 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair pathway (OGG1-BER). Accumulation of 8-oxoG in DNA has traditionally been associated with mutagenesis, as well as various human diseases and aging processes, while the free 8-oxoG base in body fluids is one of the best biomarkers of ongoing pathophysiological processes. In this review, we discuss the biological significance of the 8-oxoG base and particularly the role of OGG1-BER in the activation of small GTPases and changes in gene expression, including those that regulate pro-inflammatory chemokines/cytokines and cause inflammation.
Asunto(s)
ADN Glicosilasas/fisiología , Reparación del ADN/fisiología , Guanina/análogos & derivados , Inflamación/enzimología , Animales , Líquidos Corporales/química , Enfermedad Crónica , Citocinas/biosíntesis , Citocinas/genética , Daño del ADN , ADN Glicosilasas/deficiencia , ADN Glicosilasas/genética , Contaminantes Ambientales/toxicidad , Activación Enzimática , Células Epiteliales/enzimología , Células Epiteliales/patología , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica , Guanina/metabolismo , Humanos , Inflamación/genética , Inflamación/patología , Enfermedades Pulmonares/enzimología , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Mutagénesis , Estrés Oxidativo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Sistema Respiratorio/enzimología , Sistema Respiratorio/patologíaRESUMEN
PURPOSE: Previously we showed that the relative biological efficiency for induced cell killing by the 76-MeV beam used at the Institut Curie Proton Therapy Center in Orsay increased with depth throughout the spread-out Bragg peak (SOBP). To investigate the repair pathways underlying this increase, we used an isogenic human cell model in which individual DNA repair proteins have been depleted, and techniques dedicated to precise measurements of radiation-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). METHODS AND MATERIALS: The 3-Gy surviving fractions of HeLa cells individually depleted of Ogg1, XRCC1, and PARP1 (the base excision repair/SSB repair pathway) or of ATM, DNA-PKcs, XRCC4, and Artemis (nonhomologous end-joining pathway) were determined at the 3 positions previously defined in the SOBP. Quantification of incident SSBs and DSBs by the alkaline elution technique and 3-dimensional (3D) immunofluorescence of γ-H2AX foci, respectively, was performed in SQ20 B cells. RESULTS: We showed that the amount of SSBs and DSBs depends directly on the particle fluence and that the increase in relative biological efficiency observed in the distal part of the SOBP is due to a subset of lesions generated under these conditions, leading to cell death via a pathway in which the Artemis protein plays a central role. CONCLUSIONS: Because therapies like proton or carbon beams are now being used to treat cancer, it is even more important to dissect the mechanisms implicated in the repair of the lesions generated by these particles. Additionally, alteration of the expression or activity of the Artemis protein could be a novel therapeutic tool before high linear energy transfer irradiation treatment.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN/fisiología , Proteínas Nucleares/fisiología , Protones , Efectividad Biológica Relativa , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Instituciones Oncológicas , Supervivencia Celular/fisiología , ADN Glicosilasas/fisiología , Proteína Quinasa Activada por ADN/fisiología , Proteínas de Unión al ADN/fisiología , Endonucleasas , Francia , Células HeLa , Histonas/análisis , Humanos , Método de Montecarlo , Proteínas Nucleares/deficiencia , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/fisiología , Terapia de Protones , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Two studies by Sakurai et al. (2014) and Hu et al. (2014) in this issue of Cell Stem Cell add a new level of understanding to the mesenchymal-to-epithelial transition taking place during reprogramming, showing how this morphological transformation is promoted by Tet enzymes and blocked by kinase-dependent cytoskeletal organization.
Asunto(s)
Diferenciación Celular , Reprogramación Celular/genética , Citoesqueleto/metabolismo , ADN Glicosilasas/fisiología , Metilación de ADN , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/citología , Transición Epitelial-Mesenquimal , Células Madre Pluripotentes Inducidas/citología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/fisiología , Animales , Dioxigenasas , HumanosRESUMEN
Secondhand smoke (SHS) is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS), the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS) in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR) assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF) and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.
Asunto(s)
Daño del ADN , ADN Glicosilasas/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/fisiología , Estrés Oxidativo , Humo/efectos adversos , Secuencia de Bases , Línea Celular , Medios de Cultivo , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/citología , Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Tet-mediated DNA oxidation is a recently identified mammalian epigenetic modification, and its functional role in cell-fate transitions remains poorly understood. Here, we derive mouse embryonic fibroblasts (MEFs) deleted in all three Tet genes and examine their capacity for reprogramming into induced pluripotent stem cells (iPSCs). We show that Tet-deficient MEFs cannot be reprogrammed because of a block in the mesenchymal-to-epithelial transition (MET) step. Reprogramming of MEFs deficient in TDG is similarly impaired. The block in reprogramming is caused at least in part by defective activation of key miRNAs, which depends on oxidative demethylation promoted by Tet and TDG. Reintroduction of either the affected miRNAs or catalytically active Tet and TDG restores reprogramming in the knockout MEFs. Thus, oxidative demethylation to promote gene activation appears to be functionally required for reprogramming of fibroblasts to pluripotency. These findings provide mechanistic insight into the role of epigenetic barriers in cell-lineage conversion.