RESUMEN
The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.
Asunto(s)
Roturas del ADN de Cadena Simple , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Animales , ADN Glicosilasas/química , ADN Glicosilasas/metabolismo , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de Unión al ADN/química , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/química , Desoxirribonucleasa (Dímero de Pirimidina)/química , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Humanos , Magnesio/química , Magnesio/metabolismo , Etiquetas de Fotoafinidad/química , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
8-oxoguanine DNA glycosylase and Kin17 are proteins widely distributed and phylogenetically conserved in the CNS. 8-oxoguanine DNA glycosylase is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine present in DNA damaged by oxidative stress. Kin17 protein is involved in DNA repair and illegitimate recombination in eukaryotic cells. The present study evaluates the effect of ovarian hormones on the expression of both proteins in the magnocellular paraventricular nucleus of the hypothalamus and the bed nucleus of the stria terminalis in female and male rat brains. In the paraventricular nucleus, ovariectomy induced a significant decrease in the number of 8-oxoguanine DNA glycosylase-positive nuclei as well as in their relative fluorescent intensity as compared with ovariectomized-estradiol treated and proestrous groups. Confocal microscopy observation demonstrated that oxoguanine DNA glycosylase protein is located in the Hoechst-dyed nuclei and cytoplasm in male and ovariectomized rats. Surprisingly, following estradiol administration to ovariectomized and proestrous rats, the 8-oxoguanine DNA glycosylase immunolabeling was observed in the nucleolus, the cytoplasm and the dendrites of cells, while Kin17 protein was always localized in the cell nuclei. In the bed nucleus of the stria terminalis, the number of 8-oxoguanine DNA glycosylase-positive nuclei during proestrous was significantly lower than the number obtained in males and ovariectomized rats and similar to the number of ovariectomized-estradiol-treated groups. In contrast to these observations, no significant differences were observed in the expression of kin17 protein. Our results suggest that estrogens differentially regulate the expression of 8-oxoguanine DNA glycosylase, but not that of Kin17 protein, in specific regions of the rat brain and that estradiol can translocate the 8-oxoguanine DNA glycosylase protein within nuclei and to other subcellular compartments.
Asunto(s)
Encéfalo/metabolismo , ADN Glicosilasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Estrógenos/fisiología , Proteínas Nucleares/metabolismo , 3,3'-Diaminobencidina , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Encéfalo/citología , Recuento de Células , Núcleo Celular/metabolismo , Estradiol/farmacología , Femenino , Fluorescencia , Inmunohistoquímica , Masculino , Microscopía Confocal , Neuronas/citología , Neuronas/metabolismo , Níquel , Ovariectomía , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/metabolismo , Proestro/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Núcleos Septales/citología , Núcleos Septales/metabolismo , Distribución Tisular/efectos de los fármacosRESUMEN
The major human AP endonuclease APE1 (HAP1, APEX, Ref1) initiates the repair of abasic sites generated either spontaneously, from attack of bases by free radicals, or during the course of the repair of damaged bases. APE1 therefore plays a central role in the base excision repair (BER) pathway. We report here that XRCC1, another essential protein involved in the maintenance of genome stability, physically interacts with APE1 and stimulates its enzymatic activities. A truncated form of APE1, lacking the first 35 amino acids, although catalytically proficient, loses the affinity for XRCC1 and is not stimulated by XRCC1. Chinese ovary cell lines mutated in XRCC1 have a diminished capacity to initiate the repair of AP sites. This defect is compensated by the expression of XRCC1. XRCC1, acting as both a scaffold and a modulator of the different activities involved in BER, would provide a physical link between the incision and sealing steps of the AP site repair process. The interaction described extends the coordinating role of XRCC1 to the initial step of the repair of DNA abasic sites.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Animales , Sitios de Unión , Western Blotting , Células CHO , Catálisis , Cricetinae , ADN/metabolismo , Cartilla de ADN/química , ADN Complementario/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Factores de Tiempo , Transcripción Genética , Transfección , Técnicas del Sistema de Dos Híbridos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos XRESUMEN
Kin17 and 8-Oxoguanine DNA glycosylase (Ogg1) are proteins, respectively, involved in illegitimate recombination and DNA repair in eukaryotic cells. To characterize the expression of these proteins in cell types of rodent and avian brains, we combined immunocytochemistry for either Kin17 or Ogg1 proteins with glial fibrillary acidic protein (GFAP, an astrocyte marker) immunodetection on the same tissue section. Both Kin17 and Ogg1 proteins were localized in cell nuclei and were extensively distributed in neuronal populations of quail and rodent brains. However, GFAP-immunoreactive cells were never labeled by Kin17 protein. This was observed in nerve fiber tracts, in the cerebral cortex, the hippocampal formation, the hypothalamic region, and the periventricular regions of the brain of both species studied. These results were confirmed by combining in situ hybridization of kin17 mRNA and GFAP immunodetection. On the contrary, GFAP-immunoreactive cells were often labeled by the Ogg1 protein in brain structures such as fiber tracts, the cortical surface, the cerebellum, and the ependymal surface of both quail and mouse brains. Our results suggest that the expression of the Kin17 protein (observed in neurons) and that of the Ogg1 protein (observed in neurons and glial cells) is conserved in brain phylogeny.
Asunto(s)
Sistema Nervioso Central/enzimología , Proteínas de Unión al ADN/metabolismo , N-Glicosil Hidrolasas/metabolismo , Neuroglía/enzimología , Neuronas/enzimología , Proteínas Nucleares , Codorniz/metabolismo , Roedores/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Axones/enzimología , Axones/ultraestructura , Sistema Nervioso Central/citología , Reparación del ADN/fisiología , ADN-Formamidopirimidina Glicosilasa , Epéndimo/citología , Epéndimo/enzimología , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Ratones , Neuroglía/citología , Neuronas/citología , Codorniz/anatomía & histología , Ratas , Ratas Sprague-Dawley , Roedores/anatomía & histologíaRESUMEN
Ionizing radiation elicits a genetic response in human cells that allows cell survival. The human KIN (also known as KIN17) gene encodes a 45-kDa nuclear DNA-binding protein that participates in the response to UVC radiation and is immunologically related to the bacterial RecA protein. We report for the first time that ionizing radiation and bleomycin, a radiomimetic drug, which produce single- and double-strand breaks, increased expression of KIN in human cells established from tumors, including MeWo melanoma, MCF7 breast adenocarcinoma, and ATM+ GM3657 lymphoblast cells. KIN expression increased rapidly in a dose-dependent manner after irradiation. Under the same conditions, several genes controlled by TP53 were induced with kinetics similar to that of KIN. Using the CDKN1A gene as a marker of TP53 responsiveness, we analyzed the up-regulation of KIN and showed that is independent of the status of TP53 and ATM. In contrast, the presence of a dominant mutant for activating transcription factor 2 (ATF2) completely abolished the up-regulation of KIN. Our results suggest a role for ATF2 in the TP53-independent increase in KIN expression after gamma irradiation.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Proteínas Nucleares , Proteína p53 Supresora de Tumor/fisiología , Factor de Transcripción Activador 2 , Bleomicina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Daño del ADN , Rayos gamma , Humanos , Proteínas de Unión al ARN , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , Reparación del ADN , Guanina/análogos & derivados , N-Glicosil Hidrolasas/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Liasas de Carbono-Oxígeno/genética , Daño del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN-Formamidopirimidina Glicosilasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Guanina/química , Guanina/metabolismo , Humanos , Cinética , Mutación , N-Glicosil Hidrolasas/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión ProteicaAsunto(s)
Adenocarcinoma de Células Claras/enzimología , Neoplasias Renales/enzimología , N-Glicosil Hidrolasas/genética , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patología , ADN-Formamidopirimidina Glicosilasa , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Mutagénesis Sitio-Dirigida , N-Glicosil Hidrolasas/metabolismo , Polimorfismo Genético , Especificidad por SustratoRESUMEN
In Drosophila, the S3 ribosomal protein has been shown to act as a DNA glycosylase/AP lyase capable of releasing 8-hydroxyguanine (8-OH-Gua) in damaged DNA. Here we describe a second Drosophila protein (dOgg1) with 8-OH-Gua and abasic (AP) site DNA repair activities. The Drosophila OGG1 gene codes for a protein of 327 amino acids, which shows 33 and 37% identity with the yeast and human Ogg1 proteins, respectively. The DNA glycosylase activity of purified dOgg1 was investigated using gamma-irradiated DNA and gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The dOgg1 protein excises 8-OH-Gua and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from gamma-irradiated DNA. with k(ca)(t)/K:(M) values of 21.0 x 10(-5) and 11.2 x 10(-5) (min(-1) nM(-1)), respectively. Enzymatic assays using oligodeoxyribonucleotides containing a single lesion show that dOgg1 displays a marked preference for DNA duplexes containing 8-OH-Gua, 8-OH-Ade or an AP site placed opposite a cytosine. The cleavage of the 8-OH-Gua-containing strand results from the excision of the damaged base followed by a ss-elimination reaction at the 3'-side of the resulting AP site. Cleavage of 8-OH-Gua.C duplex involves the formation of a reaction intermediate that is converted into a stable covalent adduct in the presence of sodium borohydre. dOgg1 complements the mutator phenotype of fpg mutY mutants of Escherichia coli. Whole-mount in situ hybridizations on tissues at different stages of Drosophila development reveal that the dOGG1 messenger is expressed uniformly at a low level in cells in which mitotic division occurs. Therefore, Drosophila possesses two DNA glycosylase activities that can excise 8-OH-Gua and formamidopyrimidines from DNA, dOgg1 and the ribosomal protein S3.
Asunto(s)
Daño del ADN , Reparación del ADN , Drosophila melanogaster/genética , Proteínas de Escherichia coli , Guanina/análogos & derivados , N-Glicosil Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Catálisis , ADN/metabolismo , ADN/efectos de la radiación , ADN Glicosilasas , ADN Complementario/química , ADN Complementario/genética , ADN-Formamidopirimidina Glicosilasa , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Prueba de Complementación Genética , Guanina/metabolismo , Cinética , Larva/enzimología , Datos de Secuencia Molecular , Mutación , N-Glicosil Hidrolasas/metabolismo , Ovario/enzimología , Estrés Oxidativo , Fenotipo , Pirimidinas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The human OGG1 gene codes for a 38kD protein with an antimutator activity related to its capacity to excise the mutagenic base 8-OH-Guanine from DNA. Mutant forms of this gene have been found in lung and kidney tumors. The determination of the start of transcription allowed the definition of the promoter sequences for the gene. By transient transfection and a luciferase reporter assay a 135 base pair region immediately upstream of the transcription start is shown to have full promoter activity. Two CpG islands and an Alu repeat were identified within the promoter and the 5' sequences of the transcribed region. The lack of TATA or CAAT boxes suggests that OGG1 is a housekeeping gene. Consistently, its expression, measured as the transcription from the promoter or as the enzymatic activity in cultured fibroblast cell lines, does not vary during the cell cycle.
Asunto(s)
Ciclo Celular , Regulación de la Expresión Génica , N-Glicosil Hidrolasas/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Ciclo Celular/fisiología , ADN/análisis , ADN-Formamidopirimidina Glicosilasa , Genoma Humano , Células HeLa , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Transcripción GenéticaRESUMEN
The OGG1 gene, which codes for a DNA repair protein with antimutator activity, is located on chromosome 3p25, a frequent site of allelic deletions in many types of human tumors, including renal clear cell cancers. We present the analysis of 99 renal tumors for alterations in the OGG1 gene to determine its association with tumorigenesis. Loss of heterozygosity in the 3p25 region was found for 85% of the informative cases. We detected somatic missense mutations of the OGG1 gene in 4 of the 99 tumor samples. Biochemical analysis of the mutant proteins revealed that a substitution at codon 46 impairs the enzymatic activity. We also describe the occurrence of several polymorphisms as well as aberrantly spliced OGG1 transcripts.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Reparación del ADN/genética , Proteínas de Escherichia coli , Neoplasias Renales/genética , N-Glicosil Hidrolasas/genética , Adenocarcinoma de Células Claras/enzimología , Alelos , Cromosomas Humanos Par 3/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , ADN-Formamidopirimidina Glicosilasa , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Riñón/enzimología , Riñón/fisiología , Neoplasias Renales/enzimología , Pérdida de Heterocigocidad , Mutación Missense , N-Glicosil Hidrolasas/metabolismo , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (alpha-hOgg1-Ser(326)) (wild-type alpha-hOgg1). Mutant forms of hOgg1 with mutations Arg(46)-->Gln (alpha-hOgg1-Gln(46)) and Arg(154)-->His (alpha-hOgg1-His(154)) have previously been identified in human tumors. The mutant proteins alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type alpha-hOgg1 were investigated using gamma-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from gamma-irradiated DNA containing a multiplicity of base lesions. Michaelis-Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type alpha-hOgg1 was greater than that by alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154). The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using (32)P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with gamma-irradiated DNA. Wild-type alpha-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg(154), in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type alpha-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms alpha-hOgg1-Gln(46) and alpha-hOgg1-His(154) found in human tumors are defective in their catalytic capacities.
Asunto(s)
Proteínas de Escherichia coli , N-Glicosil Hidrolasas/metabolismo , Neoplasias/enzimología , ADN/química , ADN/genética , ADN/metabolismo , Reparación del ADN , ADN-Formamidopirimidina Glicosilasa , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Cinética , Mutación , N-Glicosil Hidrolasas/genética , Pirimidinas/metabolismo , Especificidad por SustratoRESUMEN
The oxoguanine DNA glycosylase (Ogg1) is a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine present in DNA damaged by oxidative stress. We have investigated the expression of the OGG1 gene in different regions of the rat CNS. Biochemical studies on brain homogenates of adult rats have shown that Ogg1 nicking activity is present at relatively similar levels in the cerebral cortex, the hypothalamus, the pons and the cerebellum. Following in situ hybridization with radiolabeled OGG1 cDNA or specific antisense oligonucleotides, OGG1 transcripts showed a widespread but heterogeneous distribution pattern among distinct brain regions of adult rats: high levels of this transcript were detected in the CA1-CA3 layers and the gyrus dentate of the hippocampal formation, the piriform cortex, the supraoptic nuclei, the olivary complex as well as in the pyramidal cells of layer V of the cortex and the Purkinje cells of the cerebellum. In peripheral organs such as the lungs, the stomach and the spleen, OGG1 transcript is however expressed in specific subpopulations of cells. Using a semi-quantitative reverse transcription - polymerase chain reaction assay on total mRNA from the frontal cortex, OGG1 mRNA was determined to be expressed with relatively the same levels in 1-day-old and 7-day-old rats as well as in adult rats. These results provide evidence for the widespread expression of the OGG1 gene in developing and adult brains.
Asunto(s)
Encéfalo/enzimología , Regulación Enzimológica de la Expresión Génica , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Liasas de Carbono-Oxígeno/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN-Formamidopirimidina Glicosilasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as byproducts of cell metabolism. Among DNA damages induced by ROS, 8-hydroxyguanine (8-OH-G) is certainly the product that has retained most of the attention in the past few years. The biological relevance of 8-OH-G in DNA has been unveiled by the study of Escherichia coli and Saccharomyces cerevisiae genes involved in the neutralization of the mutagenic effects of 8-OH-G. These genes, fpg and mutY for E. coli and OGG1 for yeast, code for DNA glycosylases. Inactivation of any of those genes leads to a spontaneous mutator phenotype, characterized by the increase in GC to TA transversions. In yeast, the OGG1 gene encodes a DNA glycosylase/AP lyase that excises 8-OH-G from DNA. In human cells, the OGG1 gene is localized on chromosome 3p25 and encodes two forms of hOgg1 protein which result from an alternative splicing of a single messenger RNA. The alpha-hOgg1 protein has a nuclear localization whereas the beta-hOgg1 is targeted to the mitochondrion. Biochemical studies on the alpha-hOgg1 protein show that it is a DNA glycosylase/AP lyase that excises 8-OH-G and Fapy-G from gamma-irradiated DNA. Several approaches have been used to study the biological role of OGG1 in mammalian cells, ranging from its overexpression in cell lines to the generation of homozygous ogg1-/- null mice. Furthermore, to explore a possible role in the prevention of cancer, the cDNA coding for alpha-hOgg1 has been sequenced in human tumors. All these results point to 8-OH-G as an endogenous source of mutations in eukaryotes and to its likely involvement in the process of carcinogenesis. A review of the recent literature on the mammalian Ogg1 proteins, the main repair system involved in the elimination of this mutagenic lesion, is presented.
Asunto(s)
Proteínas de Escherichia coli , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Disparidad de Par Base/genética , Secuencia de Bases , Reparación del ADN/genética , ADN-Formamidopirimidina Glicosilasa , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/deficiencia , N-Glicosil Hidrolasas/genéticaRESUMEN
The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.
Asunto(s)
Proteínas Bacterianas , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/química , Proteínas de Escherichia coli , Helicobacter pylori/enzimología , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/clasificación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Cromatografía Líquida de Alta Presión , Aductos de ADN/metabolismo , ADN Glicosilasas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Endodesoxirribonucleasas/clasificación , Inducción Enzimática , Lisina/química , Metilmetanosulfonato/farmacología , Metilnitronitrosoguanidina/farmacología , Datos de Secuencia Molecular , Mutagénesis , N-Glicosil Hidrolasas/genética , Sistemas de Lectura Abierta , Plásmidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Cigarette smoking is the major known risk factor for head and neck cancer. Tobacco promotes oxidative stress and enhances tissue levels of 8-hydroxyguanine (8-OH-G) in smokers. The presence of 8-OH-G does not impede replication but leads to an accumulation of G-->T transversions. Recently, the gene for human 8-oxoguanine DNA glycosylase 1 (hOGG1), an enzyme involved in the repair of 8-OH-G in humans, was cloned and mapped to chromosome 3p. In head and neck tumors, the hOGG1 gene locus is often targeted by loss of heterozygosity (LOH), and the spectrum of mutations in the p53 gene shows a bias in favor of G:C-->T:A transversions, as would be expected if HOGG1 repair functions were disabled. To test the involvement of hOGG1 in head and neck carcinogenesis, we had previously screened 56 tumors for LOH at 3p. From these tumors and two others, we selected 33 tumors demonstrating LOH for further mutational analysis of this gene. No somatic inactivating mutation was found in hOGG1. Polymorphisms involving intron 4 and exon 7 were present in 30% of the patients. A new polymorphism was identified in one patient in exon 6 and led to the amino-acid change G308E. Similar repair activities were found for the wild-type and exon 6-variant enzymes. Therefore, the involvement of hOGG1 in head and neck carcinogenesis is not strongly supported by this work.
Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 3 , Neoplasias de Cabeza y Cuello/genética , Pérdida de Heterocigocidad , N-Glicosil Hidrolasas/genética , Polimorfismo Genético , Alelos , Sustitución de Aminoácidos , Mapeo Cromosómico , Reparación del ADN , ADN-Formamidopirimidina Glicosilasa , Exones , Amplificación de Genes , Marcadores Genéticos , Variación Genética , Heterocigoto , Homocigoto , Humanos , IntronesRESUMEN
Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbo nyl ]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.
Asunto(s)
Daño del ADN , Mutagénesis , N-Glicosil Hidrolasas/fisiología , Animales , Bromatos/toxicidad , Línea Celular/efectos de los fármacos , Línea Celular/efectos de la radiación , Cricetinae , Cricetulus , Reparación del ADN , ADN-Formamidopirimidina Glicosilasa , Inducción Enzimática , Femenino , Genes Bacterianos , Genes Reporteros , N-Glicosil Hidrolasas/biosíntesis , N-Glicosil Hidrolasas/genética , Ovario , Oxidantes/toxicidad , Estrés Oxidativo , Fotoquímica , Fármacos Fotosensibilizantes/farmacología , Reacción en Cadena de la Polimerasa , Pirrolidinas/farmacología , Quinolizinas/farmacología , TransfecciónRESUMEN
We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation. Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS). The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N(2)O or air. Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates. Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N(2)O. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5)and 8.97 x 10(-5)(min(-1)nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326. In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326)and mutant alpha-hOgg1-Cys(326)proteins. The GST-alpha-hOgg1-Cys(326)protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326)protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA. The k (cat)/ K (m)values for excision of 8-OH-Gua and FapyGua were 2. 82 x 10(-5)and 4.43 x 10(-5)(min(-1)nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5- N -methylformamidopyrimidine (Me-FapyGua). The k (cat)/ K (m)values for excision of Me-FapyGua were 278 x 10(-5)and 319 x 10(-5)(min(-1)nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326)and GST-alpha-hOgg1-Cys(326)catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Escherichia coli , N-Glicosil Hidrolasas/metabolismo , Sustitución de Aminoácidos , Cisteína/química , Cisteína/metabolismo , ADN-Formamidopirimidina Glicosilasa , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Cromatografía de Gases y Espectrometría de Masas , Glutatión Transferasa/metabolismo , Humanos , N-Glicosil Hidrolasas/química , Estrés Oxidativo , Fenotipo , Serina/química , Serina/metabolismo , Especificidad por SustratoRESUMEN
A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as a byproduct of endogenous metabolism or the exposure to environmental oxidizing agents. An oxidatively damaged guanine, 8-hydroxyguanine (8-OH-G), is abundantly produced in DNA exposed to ROS. The biological relevance of this kind of DNA damage has been unveiled by the study of two mutator genes in E. coli, fpg and mutY. Both genes code for DNA glycosylases that cooperate to prevent the mutagenic effects of 8-OH-G. Inactivation of any of those two genes leads to a spontaneous mutator phenotype characterized by the exclusive increase in G:C to T:A transversions. In the simple eukaryote Saccharomyces cerevisiae, the OGG1 gene encodes an 8-OH-G DNA glycosylase which is the functional homolog of the bacterial fpg gene product. Moreover, the inactivation of OGG1 in yeast creates a mutator phenotype that is also specific for the generation of G:C to T:A transversions. The presence of such system in mammals has been confirmed by the cloning of the OGG1 gene coding for a human homolog of the yeast enzyme. Human cells also possess a MutY homolog encoded by the MYH gene. Analysis of the human OGG1 gene and its transcripts in normal and tumoral tissues reveals alternative splicing, polymorphisms and somatic mutations. The aim of this review is to summarize recent findings dealing with the biochemical properties and the biological functions of 8-OH-G DNA glycosylases in bacterial, yeast, insect and mammalian cells. These results point to 8-OH-G as an endogenous source of mutations and to its likely involvement in the process of carcinogenesis.
Asunto(s)
Daño del ADN , Reparación del ADN , Desoxiguanosina/análogos & derivados , Estrés Oxidativo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Transformación Celular Neoplásica/genética , Desoxiguanosina/genética , Drosophila , Escherichia coli/genética , Humanos , Mamíferos , Saccharomyces cerevisiae/genéticaRESUMEN
The human OGG1 gene encodes a DNA glycosylase activity catalysing the excision of the mutagenic lesion 7,8-dihydro-8-oxoguanine from oxidatively damaged DNA. The OGG1 gene was localized to chromosome 3p25, a region showing frequent loss of heterozygosity (LOH) in lung and kidney tumours. In this study, we have analysed by RT-PCR the expression of OGG1 in 25 small cell lung cancers, in 15 kidney carcinomas and the 15 normal kidney counterparts. The results show that OGG1 messenger RNA can be detected in all tumours tested and that no significant difference was observed in the level of expression between normal and tumoral kidney tissues. Denaturing gradient gel electrophoresis (DGGE) was used to screen this series of human tumours for alterations in the OGG1 cDNA. The study revealed homozygous mutations in three tumours, two from lung and one from kidney. Sequencing analysis of the mutants identified a single base substitution in each of the three cases: two transversions (GC to TA and TA to AT) and one transition (GC to AT). All three substitutions cause an amino acid change in the hOgg1 protein. For the mutant kidney tumour, the normal tissue counterpart shows a wild-type profile. These results suggest a role for OGG1 mutations in the course of the multistage process of carcinogenesis in lung or kidney.