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2.
J Biol Chem ; 299(3): 102991, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36758800

RESUMEN

A growing body of evidence indicates that RNA plays a critical role in orchestrating DNA double-strand break repair (DSBR). Recently, we showed that homologous nascent RNA can be used as a template for error-free repair of double-strand breaks (DSBs) in the transcribed genome and to restore the missing sequence at the break site via the transcription-coupled classical nonhomologous end-joining (TC-NHEJ) pathway. TC-NHEJ is a complex multistep process in which a reverse transcriptase (RT) is essential for synthesizing the DNA strand from template RNA. However, the identity of the RT involved in the TC-NHEJ pathway remained unknown. Here, we report that DNA polymerase eta (Pol η), known to possess RT activity, plays a critical role in TC-NHEJ. We found that Pol η forms a multiprotein complex with RNAP II and other TC-NHEJ factors, while also associating with nascent RNA. Moreover, purified Pol η, along with DSBR proteins PNKP, XRCC4, and Ligase IV can fully repair RNA templated 3'-phosphate-containing gapped DNA substrate. In addition, we demonstrate here that Pol η deficiency leads to accumulation of R-loops and persistent strand breaks in the transcribed genes. Finally, we determined that, in Pol η depleted but not in control cells, TC-NHEJ-mediated repair was severely abrogated when a reporter plasmid containing a DSB with several nucleotide deletion within the E. coli lacZ gene was introduced for repair in lacZ-expressing mammalian cells. Thus, our data strongly suggest that RT activity of Pol η is required in error-free DSBR.


Asunto(s)
Roturas del ADN de Doble Cadena , Escherichia coli , Animales , Humanos , Escherichia coli/genética , Reparación del ADN , Reparación del ADN por Unión de Extremidades , ADN , ARN/genética , ADN Ligasa (ATP) , Mamíferos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Enzimas Reparadoras del ADN/genética
3.
Nucleic Acids Res ; 49(1): 221-243, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33300026

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 ∼500 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/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(36): 22183-22192, 2020 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-32826329

RESUMEN

Among several reversible epigenetic changes occurring during transcriptional activation, only demethylation of histones and cytosine-phosphate-guanines (CpGs) in gene promoters and other regulatory regions by specific demethylase(s) generates reactive oxygen species (ROS), which oxidize DNA and other cellular components. Here, we show induction of oxidized bases and single-strand breaks (SSBs), but not direct double-strand breaks (DSBs), in the genome during gene activation by ligands of the nuclear receptor superfamily. We observed that these damages were preferentially repaired in promoters via the base excision repair (BER)/single-strand break repair (SSBR) pathway. Interestingly, BER/SSBR inhibition suppressed gene activation. Constitutive association of demethylases with BER/SSBR proteins in multiprotein complexes underscores the coordination of histone/DNA demethylation and genome repair during gene activation. However, ligand-independent transcriptional activation occurring during heat shock (HS) induction is associated with the generation of DSBs, the repair of which is likewise essential for the activation of HS-responsive genes. These observations suggest that the repair of distinct damages induced during diverse transcriptional activation is a universal prerequisite for transcription initiation. Because of limited investigation of demethylation-induced genome damage during transcription, this study suggests that the extent of oxidative genome damage resulting from various cellular processes is substantially underestimated.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo/efectos de los fármacos , Línea Celular , Islas de CpG , Roturas del ADN de Cadena Simple , Daño del ADN/efectos de los fármacos , Desmetilación , Humanos , Ligandos , ARN Mensajero , Especies Reactivas de Oxígeno
5.
Proc Natl Acad Sci U S A ; 117(14): 8154-8165, 2020 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-32205441

RESUMEN

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by CAG (encoding glutamine) repeat expansion in the Ataxin-3 (ATXN3) gene. We have shown previously that ATXN3-depleted or pathogenic ATXN3-expressing cells abrogate polynucleotide kinase 3'-phosphatase (PNKP) activity. Here, we report that ATXN3 associates with RNA polymerase II (RNAP II) and the classical nonhomologous end-joining (C-NHEJ) proteins, including PNKP, along with nascent RNAs under physiological conditions. Notably, ATXN3 depletion significantly decreased global transcription, repair of transcribed genes, and error-free double-strand break repair of a 3'-phosphate-containing terminally gapped, linearized reporter plasmid. The missing sequence at the terminal break site was restored in the recircularized plasmid in control cells by using the endogenous homologous transcript as a template, indicating ATXN3's role in PNKP-mediated error-free C-NHEJ. Furthermore, brain extracts from SCA3 patients and mice show significantly lower PNKP activity, elevated p53BP1 level, more abundant strand-breaks in the transcribed genes, and degradation of RNAP II relative to controls. A similar RNAP II degradation is also evident in mutant ATXN3-expressing Drosophila larval brains and eyes. Importantly, SCA3 phenotype in Drosophila was completely amenable to PNKP complementation. Hence, salvaging PNKP's activity can be a promising therapeutic strategy for SCA3.


Asunto(s)
Ataxina-3/genética , Reparación del ADN por Unión de Extremidades , Enzimas Reparadoras del ADN/metabolismo , Enfermedad de Machado-Joseph/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , ARN Polimerasa II/metabolismo , Proteínas Represoras/genética , Anciano de 80 o más Años , Animales , Animales Modificados Genéticamente , Ataxina-3/metabolismo , Encéfalo/patología , Línea Celular , Roturas del ADN de Doble Cadena , Modelos Animales de Enfermedad , Drosophila , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Células Madre Pluripotentes Inducidas , Enfermedad de Machado-Joseph/metabolismo , Enfermedad de Machado-Joseph/patología , Masculino , Ratones , Persona de Mediana Edad , Mutación , Péptidos/genética , ARN Interferente Pequeño/metabolismo
6.
Alzheimers Dement ; 19(4): 1245-1259, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-35993441

RESUMEN

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Animales , Humanos , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteína C9orf72/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Sistemas CRISPR-Cas , Pez Cebra/genética , Pez Cebra/metabolismo , Neuronas/metabolismo , Expansión de las Repeticiones de ADN/genética , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(10): 4696-4705, 2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30770445

RESUMEN

Genome damage and their defective repair have been etiologically linked to degenerating neurons in many subtypes of amyotrophic lateral sclerosis (ALS) patients; however, the specific mechanisms remain enigmatic. The majority of sporadic ALS patients feature abnormalities in the transactivation response DNA-binding protein of 43 kDa (TDP-43), whose nucleo-cytoplasmic mislocalization is characteristically observed in spinal motor neurons. While emerging evidence suggests involvement of other RNA/DNA binding proteins, like FUS in DNA damage response (DDR), the role of TDP-43 in DDR has not been investigated. Here, we report that TDP-43 is a critical component of the nonhomologous end joining (NHEJ)-mediated DNA double-strand break (DSB) repair pathway. TDP-43 is rapidly recruited at DSB sites to stably interact with DDR and NHEJ factors, specifically acting as a scaffold for the recruitment of break-sealing XRCC4-DNA ligase 4 complex at DSB sites in induced pluripotent stem cell-derived motor neurons. shRNA or CRISPR/Cas9-mediated conditional depletion of TDP-43 markedly increases accumulation of genomic DSBs by impairing NHEJ repair, and thereby, sensitizing neurons to DSB stress. Finally, TDP-43 pathology strongly correlates with DSB repair defects, and damage accumulation in the neuronal genomes of sporadic ALS patients and in Caenorhabditis elegans mutant with TDP-1 loss-of-function. Our findings thus link TDP-43 pathology to impaired DSB repair and persistent DDR signaling in motor neuron disease, and suggest that DSB repair-targeted therapies may ameliorate TDP-43 toxicity-induced genome instability in motor neuron disease.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/genética , Humanos , Neuronas Motoras/metabolismo , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo
8.
Int J Mol Sci ; 23(9)2022 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-35563044

RESUMEN

The damage and repair of DNA is a continuous process required to maintain genomic integrity. DNA double-strand breaks (DSBs) are the most lethal type of DNA damage and require timely repair by dedicated machinery. DSB repair is uniquely important to nondividing, post-mitotic cells of the central nervous system (CNS). These long-lived cells must rely on the intact genome for a lifetime while maintaining high metabolic activity. When these mechanisms fail, the loss of certain neuronal populations upset delicate neural networks required for higher cognition and disrupt vital motor functions. Mammalian cells engage with several different strategies to recognize and repair chromosomal DSBs based on the cellular context and cell cycle phase, including homologous recombination (HR)/homology-directed repair (HDR), microhomology-mediated end-joining (MMEJ), and the classic non-homologous end-joining (NHEJ). In addition to these repair pathways, a growing body of evidence has emphasized the importance of DNA damage response (DDR) signaling, and the involvement of heterogeneous nuclear ribonucleoprotein (hnRNP) family proteins in the repair of neuronal DSBs, many of which are linked to age-associated neurological disorders. In this review, we describe contemporary research characterizing the mechanistic roles of these non-canonical proteins in neuronal DSB repair, as well as their contributions to the etiopathogenesis of selected common neurological diseases.


Asunto(s)
Roturas del ADN de Doble Cadena , Enfermedades del Sistema Nervioso , Animales , ADN/genética , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Mamíferos/genética , Enfermedades del Sistema Nervioso/genética , Reparación del ADN por Recombinación
9.
Hum Mol Genet ; 28(15): 2459-2476, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31067307

RESUMEN

Dominant mutations in the RNA/DNA-binding protein TDP-43 have been linked to amyotrophic lateral sclerosis (ALS). Here, we screened genomic DNA extracted from spinal cord specimens of sporadic ALS patients for mutations in the TARDBP gene and identified a patient specimen with previously reported Q331K mutation. The patient spinal cord tissue with Q331K mutation showed accumulation of higher levels of DNA strand breaks and the DNA double-strand break (DSB) marker γH2AX, compared to age-matched controls, suggesting a role of the Q331K mutation in genome-damage accumulation. Using conditional SH-SY5Y lines ectopically expressing wild-type (WT) or Q331K-mutant TDP-43, we confirmed the increased cytosolic sequestration of the poly-ubiquitinated and aggregated form of mutant TDP-43, which correlated with increased genomic DNA strand breaks, activation of the DNA damage response factors phospho-ataxia-telangiectasia mutated (ATM), phospho-53BP1, γH2AX and neuronal apoptosis. We recently reported the involvement of WT TDP-43 in non-homologous end joining (NHEJ)-mediated DSB repair, where it acts as a scaffold for the recruitment of XRCC4-DNA ligase 4 complex. Here, the mutant TDP-43, due to its reduced interaction and enhanced cytosolic mislocalization, prevented the nuclear translocation of XRCC4-DNA ligase 4. Consistently, the mutant cells showed significantly reduced DNA strand break sealing activity and were sensitized to DNA-damaging drugs. In addition, the mutant cells showed elevated levels of reactive oxygen species, suggesting both dominant negative and loss-of-function effects of the mutation. Together, our study uncovered an association of sporadic Q331K mutation with persistent genome damage accumulation due to both damage induction and repair defects.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , ADN Ligasa (ATP)/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación , Esclerosis Amiotrófica Lateral/metabolismo , Apoptosis/genética , Línea Celular , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP)/genética , Reparación del ADN , Humanos , Neuronas/metabolismo , Polimorfismo de Nucleótido Simple , Proteínas de Unión al ARN/metabolismo , Médula Espinal/metabolismo , Translocación Genética
10.
Cell Mol Life Sci ; 76(4): 729-743, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30374520

RESUMEN

Cannabinoids are the most commonly abused illicit drugs worldwide. While cannabis can be beneficial for certain heath conditions, abuse of potent synthetic cannabinoids has been on the rise. Exposure to cannabinoids is also prevalent in women of child-bearing age and pregnant women. These compounds can cross the placental barrier and directly affect the fetus. They mediate their effects primarily through G-protein coupled cannabinoid receptors, CB1 and CB2. In addition to significant neurological effects, cannabinoids can trigger robust immunomodulation by altering cytokine levels, causing apoptosis of lymphoid cells and inducing suppressor cells of the immune system. Profound effects of cannabinoids on the immune system as discussed in this review, suggest that maternal exposure during pregnancy could lead to dysregulation of innate and adaptive immune system of developing fetus and offspring potentially leading to weakening of immune defenses against infections and cancer later in life. Emerging evidence also indicates the underlying role of epigenetic mechanisms causing long-lasting impact following cannabinoid exposure in utero.


Asunto(s)
Cannabinoides/envenenamiento , Desarrollo Fetal/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Inmunidad Adaptativa/inmunología , Cannabinoides/química , Femenino , Desarrollo Fetal/inmunología , Humanos , Sistema Inmunológico/embriología , Sistema Inmunológico/inmunología , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Estructura Molecular , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente
11.
Nucleic Acids Res ; 45(5): 2585-2599, 2017 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-27994036

RESUMEN

Microhomology-mediated end joining (MMEJ), an error-prone pathway for DNA double-strand break (DSB) repair, is implicated in genomic rearrangement and oncogenic transformation; however, its contribution to repair of radiation-induced DSBs has not been characterized. We used recircularization of a linearized plasmid with 3΄-P-blocked termini, mimicking those at X-ray-induced strand breaks, to recapitulate DSB repair via MMEJ or nonhomologous end-joining (NHEJ). Sequence analysis of the circularized plasmids allowed measurement of relative activity of MMEJ versus NHEJ. While we predictably observed NHEJ to be the predominant pathway for DSB repair in our assay, MMEJ was significantly enhanced in preirradiated cells, independent of their radiation-induced arrest in the G2/M phase. MMEJ activation was dependent on XRCC1 phosphorylation by casein kinase 2 (CK2), enhancing XRCC1's interaction with the end resection enzymes MRE11 and CtIP. Both endonuclease and exonuclease activities of MRE11 were required for MMEJ, as has been observed for homology-directed DSB repair (HDR). Furthermore, the XRCC1 co-immunoprecipitate complex (IP) displayed MMEJ activity in vitro, which was significantly elevated after irradiation. Our studies thus suggest that radiation-mediated enhancement of MMEJ in cells surviving radiation therapy may contribute to their radioresistance and could be therapeutically targeted.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Humanos , Fosforilación , Rayos X , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X
12.
Nucleic Acids Res ; 45(2): 739-748, 2017 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-27794043

RESUMEN

Reactive oxygen species (ROS), generated both endogenously and in response to exogenous stress, induce point mutations by mis-replication of oxidized bases and other lesions in the genome. Repair of these lesions via base excision repair (BER) pathway maintains genomic fidelity. Regulation of the BER pathway for mutagenic oxidized bases, initiated by NEIL1 and other DNA glycosylases at the chromatin level remains unexplored. Whether single nucleotide (SN)-BER of a damaged base requires histone deposition or nucleosome remodeling is unknown, unlike nucleosome reassembly which is shown to be required for other DNA repair processes. Here we show that chromatin assembly factor (CAF)-1 subunit A (CHAF1A), the p150 subunit of the histone H3/H4 chaperone, and its partner anti-silencing function protein 1A (ASF1A), which we identified in human NEIL1 immunoprecipitation complex, transiently dissociate from chromatin bound NEIL1 complex in G1 cells after induction of oxidative base damage. CHAF1A inhibits NEIL1 initiated repair in vitro Subsequent restoration of the chaperone-BER complex in cell, presumably after completion of repair, suggests that histone chaperones sequester the repair complex for oxidized bases in non-replicating chromatin, and allow repair when oxidized bases are induced in the genome.


Asunto(s)
Factor 1 de Ensamblaje de la Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Oxidación-Reducción , Estrés Oxidativo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Daño del ADN/efectos de la radiación , ADN Glicosilasas/metabolismo , Glucosa Oxidasa/metabolismo , Histonas/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos , Unión Proteica , Radiación Ionizante , Especies Reactivas de Oxígeno , Factores de Transcripción
13.
Int J Mol Sci ; 20(22)2019 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-31703296

RESUMEN

Alzheimer's disease (AD) is the most devastating neurodegenerative disorder that affects the aging population worldwide. Endogenous and exogenous factors are involved in triggering this complex and multifactorial disease, whose hallmark is Amyloid-ß (Aß), formed by cleavage of amyloid precursor protein by ß- and γ-secretase. While there is no definitive cure for AD to date, many neuroprotective natural products, such as polyphenol and carotenoid compounds, have shown promising preventive activity, as well as helping in slowing down disease progression. In this article, we focus on the chemistry as well as structure of carotenoid compounds and their neuroprotective activity against Aß aggregation using molecular docking analysis. In addition to examining the most prevalent anti-amyloidogenic carotenoid lutein, we studied cryptocapsin, astaxanthin, fucoxanthin, and the apocarotenoid bixin. Our computational structure-based drug design analysis and molecular docking simulation revealed important interactions between carotenoids and Aß via hydrogen bonding and van der Waals interactions, and shows that carotenoids are powerful anti-amyloidogenic molecules with a potential role in preventing AD, especially since most of them can cross the blood-brain barrier and are considered nutraceutical compounds. Our studies thus illuminate mechanistic insights on how carotenoids inhibit Aß aggregation. The potential role of carotenoids as novel therapeutic molecules in treating AD and other neurodegenerative disorders are discussed.


Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Carotenoides , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Carotenoides/química , Carotenoides/uso terapéutico , Humanos , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/uso terapéutico
14.
PLoS Genet ; 11(1): e1004749, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25633985

RESUMEN

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.


Asunto(s)
Enzimas Reparadoras del ADN/genética , Enfermedad de Machado-Joseph/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Represoras/genética , Expansión de Repetición de Trinucleótido/genética , Animales , Ataxina-3 , Línea Celular , Daño del ADN/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Humanos , Enfermedad de Machado-Joseph/enzimología , Enfermedad de Machado-Joseph/fisiopatología , Mamíferos , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Estrés Oxidativo/genética , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Represoras/metabolismo
15.
J Biol Chem ; 291(49): 25553-25566, 2016 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-27756845

RESUMEN

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression.


Asunto(s)
ADN Glicosilasas/biosíntesis , Epigénesis Genética , Regulación Enzimológica de la Expresión Génica , Guanina/análogos & derivados , FN-kappa B/metabolismo , Elementos de Respuesta , Animales , ADN Glicosilasas/genética , Reparación del ADN , Guanina/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , FN-kappa B/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
16.
J Biol Chem ; 290(34): 20919-20933, 2015 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-26134572

RESUMEN

The human DNA glycosylase NEIL1 was recently demonstrated to initiate prereplicative base excision repair (BER) of oxidized bases in the replicating genome, thus preventing mutagenic replication. A significant fraction of NEIL1 in cells is present in large cellular complexes containing DNA replication and other repair proteins, as shown by gel filtration. However, how the interaction of NEIL1 affects its recruitment to the replication site for prereplicative repair was not investigated. Here, we show that NEIL1 binarily interacts with the proliferating cell nuclear antigen clamp loader replication factor C, DNA polymerase δ, and DNA ligase I in the absence of DNA via its non-conserved C-terminal domain (CTD); replication factor C interaction results in ∼8-fold stimulation of NEIL1 activity. Disruption of NEIL1 interactions within the BERosome complex, as observed for a NEIL1 deletion mutant (N311) lacking the CTD, not only inhibits complete BER in vitro but also prevents its chromatin association and reduced recruitment at replication foci in S phase cells. This suggests that the interaction of NEIL1 with replication and other BER proteins is required for efficient repair of the replicating genome. Consistently, the CTD polypeptide acts as a dominant negative inhibitor during in vitro repair, and its ectopic expression sensitizes human cells to reactive oxygen species. We conclude that multiple interactions among BER proteins lead to large complexes, which are critical for efficient BER in mammalian cells, and the CTD interaction could be targeted for enhancing drug/radiation sensitivity of tumor cells.


Asunto(s)
ADN Glicosilasas/genética , Reparación del ADN , Replicación del ADN , Genoma Humano , Secuencia de Bases , Daño del ADN , ADN Glicosilasas/deficiencia , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Datos de Secuencia Molecular , Estrés Oxidativo , Estructura Terciaria de Proteína , Radiación Ionizante , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicación C , Fase S/genética , Fase S/efectos de la radiación , Transducción de Señal
18.
Cell Mol Life Sci ; 72(9): 1679-98, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25575562

RESUMEN

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory proteins guiding distinct BER sub-pathways.


Asunto(s)
Daño del ADN , Reparación del ADN , ADN/genética , Genoma Humano , Estrés Oxidativo , ADN/química , ADN/metabolismo , ADN Glicosilasas/metabolismo , Replicación del ADN , Humanos , Mutación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Mapas de Interacción de Proteínas , Proteínas de Unión al ARN/metabolismo , Transcripción Genética
19.
Proc Natl Acad Sci U S A ; 110(33): E3090-9, 2013 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-23898192

RESUMEN

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a "cowcatcher" and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function.


Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN/genética , Replicación del ADN/fisiología , Genoma Humano/genética , Estrés Oxidativo/fisiología , Western Blotting , Bromodesoxiuridina , Inmunoprecipitación de Cromatina , ADN Polimerasa III/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Microscopía Fluorescente , Estrés Oxidativo/genética , ARN Interferente Pequeño/genética
20.
Int J Mol Sci ; 15(9): 16975-97, 2014 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-25250913

RESUMEN

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/patología
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