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1.
Nucleic Acids Res ; 44(11): 5190-203, 2016 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-26980281

RESUMEN

DNA trinucleotide repeat (TNR) expansion underlies several neurodegenerative disorders including Huntington's disease (HD). Accumulation of oxidized DNA bases and their inefficient processing by base excision repair (BER) are among the factors suggested to contribute to TNR expansion. In this study, we have examined whether oxidation of the purine dNTPs in the dNTP pool provides a source of DNA damage that promotes TNR expansion. We demonstrate that during BER of 8-oxoguanine (8-oxodG) in TNR sequences, DNA polymerase ß (POL ß) can incorporate 8-oxodGMP with the formation of 8-oxodG:C and 8-oxodG:A mispairs. Their processing by the OGG1 and MUTYH DNA glycosylases generates closely spaced incisions on opposite DNA strands that are permissive for TNR expansion. Evidence in HD model R6/2 mice indicates that these DNA glycosylases are present in brain areas affected by neurodegeneration. Consistent with prevailing oxidative stress, the same brain areas contained increased DNA 8-oxodG levels and expression of the p53-inducible ribonucleotide reductase. Our in vitro and in vivo data support a model where an oxidized dNTPs pool together with aberrant BER processing contribute to TNR expansion in non-replicating cells.


Asunto(s)
ADN Glicosilasas/metabolismo , ADN/genética , ADN/metabolismo , Inestabilidad Genómica , Repeticiones de Trinucleótidos , 8-Hidroxi-2'-Desoxicoguanosina , Animales , ADN Polimerasa beta/metabolismo , Reparación del ADN , Desoxiguanosina/análogos & derivados , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Oxidación-Reducción , Estrés Oxidativo , Expansión de Repetición de Trinucleótido
2.
Nucleic Acids Res ; 41(7): 4093-103, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23460202

RESUMEN

The MUTYH DNA-glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA-glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1:HUS1 (9-1-1) complex. In comparison with Mutyh(-)(/)(-) mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9-1-1 complex leads to a repair-defective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.


Asunto(s)
ADN Glicosilasas/fisiología , Reparación del ADN , Sustitución de Aminoácidos , Animales , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Ratones , Ratones Noqueados , Mutación , Estrés Oxidativo , Proteínas Recombinantes/metabolismo
3.
Neurobiol Dis ; 49: 148-58, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22974734

RESUMEN

Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (Hdh(Q111)). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of Hdh(Q111) cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals.


Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Enfermedad de Huntington/fisiopatología , Mitocondrias/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular , Células Cultivadas , ADN Mitocondrial/metabolismo , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Peróxido de Hidrógeno/toxicidad , Potencial de la Membrana Mitocondrial/fisiología , Ratones Transgénicos , Mitocondrias/patología , Músculo Esquelético/metabolismo , Mutación , Miocardio/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Nitrocompuestos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Propionatos/toxicidad , Especies Reactivas de Oxígeno/metabolismo
4.
PLoS Genet ; 4(11): e1000266, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19023407

RESUMEN

Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG) in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntington's disease. hMTH1 transgene expression conferred a dramatic protection against these Huntington's disease-like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntington's disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntington's disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.


Asunto(s)
Cuerpo Estriado/metabolismo , Guanina/análogos & derivados , Enfermedad de Huntington/metabolismo , Enfermedades Neurodegenerativas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Daño del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , ADN Complementario/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Guanina/metabolismo , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/genética , Nitrocompuestos/toxicidad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Propionatos/toxicidad , Células Madre/metabolismo
5.
Mutat Res ; 703(1): 59-65, 2010 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-20601098

RESUMEN

Oxidative DNA damage can be the consequence of endogenous metabolic processes and exogenous insults and several DNA repair enzymes provide protection against the toxic effects of oxidized DNA bases. Here we review the increasing knowledge on the relationship between an oxidized dNTPs pool and genome instability. The review also describes some important progress toward understanding the role of oxidative DNA damage and its repair in neurodegenerative diseases. In particular the hMTH1 hydrolase destroys oxidized nucleic acid precursors to prevent their harmful incorporation into DNA and RNA. Based on results obtained in our transgenic mouse overexpressing hMTH1 in the brain we discussed the mechanisms by which this hydrolase protects against neurodegeneration in Huntington disease models.


Asunto(s)
Nucleótidos de Desoxiguanina/metabolismo , Inestabilidad Genómica , Enfermedades Neurodegenerativas/metabolismo , Nucleótidos de Purina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Daño del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Humanos , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/metabolismo
6.
Cancer Res ; 66(18): 9036-44, 2006 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-16982745

RESUMEN

We describe a new approach to investigate alterations in the human MLH1 mismatch repair (MMR) gene. This is based on complementation of the phenotype of a MLH1-defective subclone of the ovarian carcinoma A2780 cells by transfection of vectors encoding altered MLH1 proteins. Measurements of resistance (tolerance) to methylating agents, mutation rate at HPRT, microsatellite instability (MSI), and steady-state levels of DNA 8-oxoguanine were used to define the MMR status of transfected clones. The approach was validated by transfecting cDNA of wild-type (WT) MLH1, cDNAs bearing two previously identified polymorphisms (I219V and I219L) and two with confirmed hereditary nonpolyposis colorectal cancer (HNPCC) syndrome mutations (G224D and G67R). A low-level expression of two MLH1 polymorphisms partially reversed methylation tolerance and the mutator phenotype, including MSI. Higher levels of I219V resulted in full restoration of these properties to WT. Increased expression of I129L did not fully complement the MLH1 defect, because there was a simultaneous escalation in the level of oxidative DNA damage. The findings confirmed the important relationship between deficient MMR and increased levels of oxidative DNA damage. Mutations from Italian HNPCC families (G224D, G67R, N635S, and K618A) were all ineffective at reversing the phenotype of the MLH1-defective A2780 cells. One (K618A) was identified as a low penetrance mutation based on clinical and genetic observations.


Asunto(s)
Proteínas Portadoras/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Reparación del ADN/genética , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Proteínas Adaptadoras Transductoras de Señales , Disparidad de Par Base , Proteínas Portadoras/biosíntesis , Línea Celular Tumoral , Daño del ADN , ADN Complementario/genética , Femenino , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Homólogo 1 de la Proteína MutL , Mutación Missense , Proteínas Nucleares/biosíntesis , Polimorfismo Genético , Transfección
7.
Nat Commun ; 5: 4990, 2014 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-25241845

RESUMEN

Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant. Here we studied the phenotype of variants of the essential replicative polymerase-δ carrying missense mutations in its active site, similar to those recently identified in familial colon cancer patients. We show that expression of the mutants but not the wild-type protein endows the engineered cells with a mutator phenotype and that the mutations affect the fidelity and/or the exonuclease activity of the isolated enzyme in vitro. This proof-of-principle study demonstrates the general applicability of this experimental approach in the study of genotype-phenotype correlations.


Asunto(s)
ADN Polimerasa III/genética , Mutación Missense , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Neoplasias del Colon/genética , ADN Complementario/metabolismo , Citometría de Flujo , Estudios de Asociación Genética , Vectores Genéticos , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Fenotipo , Interferencia de ARN , Transfección
8.
Aging Cell ; 12(4): 695-705, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23648059

RESUMEN

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.


Asunto(s)
Conducta Animal , Enzimas Reparadoras del ADN/metabolismo , Conducta Exploratoria , Regulación del Desarrollo de la Expresión Génica , Longevidad , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Senescencia Celular , Enzimas Reparadoras del ADN/genética , Femenino , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/genética , Factores de Tiempo
9.
DNA Repair (Amst) ; 11(6): 579-86, 2012 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-22487424

RESUMEN

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase κ (pol κ) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol κ has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol κ is down-regulated was affected by the levels of O6-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol κ status. The downregulation of pol κ caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O6-methylguanine ( O6-meGua) lesions. After MNU exposure, in the absence of pol κ, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol κ partially protects human cells from the MMR-dependent cytotoxicity of O6-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O6-meGua facilitated by RAD 51 binding.


Asunto(s)
Reparación de la Incompatibilidad de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/análogos & derivados , Alquilantes/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , ADN Polimerasa Dirigida por ADN/genética , Guanina/metabolismo , Células HeLa , Humanos , Immunoblotting , Metilnitrosourea/farmacología , Mitosis/efectos de los fármacos , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Interferencia de ARN , Recombinasa Rad51/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambio de Cromátides Hermanas/efectos de los fármacos , Rayos Ultravioleta
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