RESUMEN
Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."
Asunto(s)
Daño del ADN , ADN Mitocondrial/genética , Daño por Reperfusión Miocárdica/genética , Animales , Peso Corporal , ADN Glicosilasas/deficiencia , Reparación del ADN , Modelos Animales de Enfermedad , Dosificación de Gen , Masculino , Ratones , Ratones Noqueados , Daño por Reperfusión Miocárdica/patología , Fenotipo , Factores de TiempoRESUMEN
The base excision repair pathway has evolved to protect cells from the deleterious effects of endogenous DNA damage induced by hydrolysis, reactive oxygen species and other intracellular metabolites that modify DNA base structure. However, base excision repair is also important to resist lesions produced by ionizing radiation and strong alkylating agents, which are similar to those induced by endogenous factors.
Asunto(s)
Reparación del ADN , Modelos Genéticos , Adenina , Alquilación , Secuencia de Aminoácidos , Composición de Base , Citosina , Daño del ADN , Endodesoxirribonucleasas , Hidrólisis , Datos de Secuencia Molecular , N-Glicosil Hidrolasas , Purinas , UraciloRESUMEN
Endonuclease III from Escherichia coli is the prototype of a ubiquitous DNA repair enzyme essential for the removal of oxidized pyrimidine base damage. The yeast genome project has revealed the presence of two genes in Saccharomyces cerevisiae, NTG1 and NTG2, encoding proteins with similarity to endonuclease III. Both contain the highly conserved helix-hairpin-helix motif, whereas only one (Ntg2) harbors the characteristic iron-sulfur cluster of the endonuclease III family. We have characterized these gene functions by mutant and enzyme analysis as well as by gene expression and intracellular localization studies. Targeted gene disruption of NTG1 and NTG2 produced mutants with greatly increased spontaneous and hydrogen peroxide-induced mutation frequency relative to the wild type, and the mutation response was further increased in the double mutant. Both enzymes were found to remove thymine glycol and 2, 6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (faPy) residues from DNA with high efficiency. However, on UV-irradiated DNA, saturating concentrations of Ntg2 removed only half of the cytosine photoproducts released by Ntg1. Conversely, 5-hydroxycytosine was removed efficiently only by Ntg2. The enzymes appear to have different reaction modes, as judged from much higher affinity of Ntg2 for damaged DNA and more efficient borhydride trapping of Ntg1 to abasic sites in DNA despite limited DNA binding. Northern blot and promoter fusion analysis showed that NTG1 is inducible by cell exposure to DNA-damaging agents, whereas NTG2 is constitutively expressed. Ntg2 appears to be a nuclear enzyme, whereas Ntg1 was sorted both to the nucleus and to the mitochondria. We conclude that functions of both NTG1 and NTG2 are important for removal of oxidative DNA damage in yeast.
Asunto(s)
Daño del ADN/genética , Reparación del ADN/efectos de los fármacos , Desoxirribonucleasa (Dímero de Pirimidina) , Proteínas de Escherichia coli , N-Glicosil Hidrolasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Citosina/análogos & derivados , Citosina/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Endodesoxirribonucleasas/metabolismo , Escherichia coli/enzimología , Regulación Fúngica de la Expresión Génica/genética , Marcación de Gen , Genes Fúngicos/genética , Secuencias Hélice-Asa-Hélice/genética , Peróxido de Hidrógeno/farmacología , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación/genética , Estrés Oxidativo , Pirimidinas/metabolismo , ARN Mensajero/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Timina/análogos & derivados , Rayos UltravioletaRESUMEN
The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.
Asunto(s)
Reparación del ADN , ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , N-Glicosil Hidrolasas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Anisotropía , ADN/química , ADN/genética , Daño del ADN , ADN-Formamidopirimidina Glicosilasa , Desoxiguanosina/genética , Polarización de Fluorescencia/métodos , Humanos , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismoRESUMEN
The quality of germ cell DNA is critical for the fate of the offspring, yet there is limited knowledge of the DNA repair capabilities of such cells. One of the main DNA repair pathways is base excision repair (BER) which is initiated by DNA glycosylases that excise damaged bases, followed by incision of the generated abasic (AP) sites. We have studied human and rat methylpurine-DNA glycosylase (MPG), uracil-DNA glycosylase (UNG), and the major AP endonuclease (HAP1/APEX) in male germ cells. Enzymatic activities and western analyses indicate that these enzymes are present in human and rat male germ cells in amounts that are at least as high as in somatic cells. Minor differences were observed between different cellular stages of rat spermatogenesis and spermiogenesis. Repair of methylated DNA was also studied at the cellular level using the Comet assay. The repair was highly efficient in both human and rat male germ cells, in primary spermatocytes as well as round spermatids, compared to rat mononuclear blood cells or hepatocytes. This efficient BER removes frequently occurring DNA lesions that arise spontaneously or via environmental agents, thereby minimising the number of potential mutations transferred to the next generation.
Asunto(s)
Reparación del ADN/genética , Espermatozoides/metabolismo , Animales , Células Sanguíneas/metabolismo , Western Blotting , Liasas de Carbono-Oxígeno/metabolismo , Extractos Celulares , Tamaño de la Célula , Células Cultivadas , Ensayo Cometa , ADN/genética , ADN/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , ADN Glicosilasas , Metilación de ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Hepatocitos/metabolismo , Humanos , Masculino , Meiosis/genética , Metilmetanosulfonato/farmacología , Mutación/genética , N-Glicosil Hidrolasas/metabolismo , Ratas , Espermátides/citología , Espermátides/efectos de los fármacos , Espermátides/enzimología , Espermátides/metabolismo , Espermatogénesis/genética , Espermatozoides/citología , Espermatozoides/efectos de los fármacos , Espermatozoides/enzimología , Uracilo/metabolismo , Uracil-ADN GlicosidasaRESUMEN
In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.
Asunto(s)
Reparación del ADN , Guanina/metabolismo , N-Glicosil Hidrolasas/metabolismo , Secuencia de Bases , Liasas de Carbono-Oxígeno/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa beta/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/metabolismo , ADN-Formamidopirimidina Glicosilasa , Endodesoxirribonucleasas/metabolismo , Endonucleasas de ADN Solapado , Guanina/análogos & derivados , Humanos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación CRESUMEN
Inducible expression of the mammalian glial cell glutamate transporter GLT-1 has been established in a CHO cell line selected for low endogenous Na+-dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline-containing medium, to activate GLT-1 expression via the Tet-On system, increased uptake of the GLT-1 substrate D-aspartate 280-fold, and increased cell size. Applying glutamate to whole-cell clamped, doxycycline-treated cells evoked a transporter-mediated current with characteristics appropriate for GLT-1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT-1, the most abundant glutamate transporter in the brain.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/biosíntesis , Ácido Glutámico/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico , Células CHO , Tamaño de la Célula , Cricetinae , Doxiciclina/farmacología , Sodio/metabolismo , TransfecciónRESUMEN
Glutamate is the major excitatory transmitter in the mammalian central nervous system. Glutamate transporters, which keep the extracellular glutamate concentration low, are required both for normal brain function and for protecting neurons against harmful glutamatergic overstimulation. We have isolated the cDNA for a rat brain glutamate transporter (REAAC1) which has 90% amino acid and 86% nucleotide identity to the rabbit EAAC1. When REAAC1 was expressed in HeLa cells using a recombinant vaccinia-T7 virus expression system, a sodium dependent glutamate uptake was observed. The affinity of the carrier to various substrates was typical of brain "high affinity' glutamate uptake: threo-3-hydroxyaspartate, (R)-aspartate, (S)-glutamate and (S)-trans-pyrrolidine-2,4-dicarboxylic acid were strong inhibitors, but not (R)-glutamate or gamma-aminobutyrate. High resolution, non-radioactive in situ hybridization histochemistry in rat brain revealed the mRNA in several types of glutamatergic as well as non-glutamatergic neurons, but not in glial cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Cerebelo/citología , ADN Complementario/aislamiento & purificación , Neuronas/metabolismo , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico , Células Cultivadas , Clonación Molecular , Células HeLa , Humanos , Datos de Secuencia Molecular , RatasRESUMEN
The systemic Neisseria meningitidis strain P241 and the healthy pharyngeal carrier strain BT878 produce bacteriocin-like substances during growth. A method has been devised for obtaining the active substances in solution. The activity was recovered by freeze-thaw extraction of dialyzed Todd-Hewitt agar medium into which the bacteriocins had diffused during growth of the producer strains. The bacteriocins were purified more than 50-fold by ammonium-sulphate precipitation and hydrophobic interaction chromatography. They are quite stable to heat and remain active 100% after 30 min at 100 degrees C. However, the protein nature of the bacteriocins has been confirmed by their sensitivity to alpha-chymotrypsin. Gel filtration indicated an Mr of 100-110 kDa, whereas SDS-polyacrylamide gel electrophoresis produced a common band by Coomassie staining corresponding to an Mr of 47-48 kDa, suggesting a dimer form of the active protein component.
Asunto(s)
Bacteriocinas/biosíntesis , Neisseria meningitidis/metabolismo , Bacteriocinas/química , Bacteriocinas/aislamiento & purificación , Humanos , Peso MolecularRESUMEN
Endonuclease III (Nth) of Escherichia coli is a DNA glycosylase essential for the removal of oxidised pyrimidine base residues from DNA. Several eukaryotic homologues have recently been identified and shown to have biochemical properties similar to those of Nth. However, some of the eukaryotic counterparts also appear to remove imidazole ring-opened purine residues (faPy), a property not shared by the enzymes of bacterial origin. Here, we show that the human enzyme also possesses efficient faPy DNA glycosylase activity as indicated both from studies of the purified protein and induced overexpression of the human NTH1 cDNA in HeLa cells. We constructed green fluorescent protein-tagged hNTH1 fusion proteins to study the cellular localisation of hNTH1 and found strong and exclusive sorting to the nucleus. Studies with synchronised cells showed that the expression of hNTH1 is regulated during the cell cycle with increased transcription during early and mid S-phase.
Asunto(s)
Ciclo Celular , Reparación del ADN/genética , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Regulación Enzimológica de la Expresión Génica , N-Glicosil Hidrolasas/metabolismo , Pirimidinas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Núcleo Celular/metabolismo , Clonación Molecular , ADN-Formamidopirimidina Glicosilasa , Doxiciclina/farmacología , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Queratinocitos/enzimología , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/aislamiento & purificación , Señales de Localización Nuclear/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Fase S/genéticaRESUMEN
Chlorinated tap water often contains 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), which is a potent directly acting bacterial mutagen. We have investigated the induction of DNA damage by MX in a promyelocytic human leukaemia cell line (HL-60 cells). Exposure of HL-60 cells to 100-300 microM MX resulted in increased levels of DNA single-strand breaks and/or alkali-labile sites (SSBs) as detected by alkaline filter elution. When adding inhibitors of DNA break repair (AraC plus hydroxyurea), increased levels of DNA SSBs were observed at very low concentrations (1-3 microM) of MX, as observed by both alkaline filter elution and the single-cell gel electrophoresis assay. Increased DNA SSBs could also be observed if DNA repair inhibitors were added immediately after exposure to 10 microM MX, indicating that low concentrations of MX cause a relatively stable modification of DNA that may be recognized and incised by DNA repair enzyme activities. Further studies with DNA break repair inhibitors indicated that HL-60 cells exposed to 10 microM MX for 1 h repaired 50% of their initial DNA damage during a 2-h period and the repair appeared to be complete at 22 h. Analysis of MX-treated DNA by sequencing methods indicated that MX preferentially reacts with guanines in DNA.
Asunto(s)
Daño del ADN , ADN/química , Furanos/toxicidad , Células HL-60/efectos de los fármacos , Citarabina/farmacología , ADN/efectos de los fármacos , ADN de Cadena Simple/química , ADN de Cadena Simple/efectos de los fármacos , Electroforesis/métodos , Filtración , Furanos/química , Guanina/química , Guanina/metabolismo , Humanos , Hidroxiurea/farmacología , Mutágenos/toxicidad , Análisis de Secuencia de ADNRESUMEN
Genome alterations due to horizontal gene transfer and stress constantly generate strain on the gene pool of Neisseria meningitidis, the causative agent of meningococcal (MC) disease. The DNA glycosylase MutY of the base excision repair pathway is involved in the protection against oxidative stress. MC MutY expressed in Escherichia coli exhibited base excision activity towards DNA substrates containing A:7,8-dihydro-8-oxo-2'-deoxyguanosine and A:C mismatches. Expression in E. coli fully suppressed the elevated spontaneous mutation rate found in the E. coli mutY mutant. An assessment of MutY activity in lysates of neisserial wild-type and mutY mutant strains showed that both MC and gonococcal (GC) MutY is expressed and active in vivo. Strikingly, MC and GC mutY mutants exhibited 60- to 140-fold and 20-fold increases in mutation rates, respectively, compared to the wild-type strains. Moreover, the differences in transitions and transversions in rpoB conferring rifampin resistance observed with the wild type and mutants demonstrated that the neisserial MutY enzyme works in preventing GC-->AT transversions. These findings are important in the context of models linking mutator phenotypes of disease isolates to microbial fitness.
Asunto(s)
Disparidad de Par Base/genética , ADN Glicosilasas/metabolismo , Guanina/metabolismo , Neisseria/enzimología , ADN Glicosilasas/genética , ADN Glicosilasas/fisiología , Análisis Mutacional de ADN , Reparación del ADN , Guanina/análogos & derivados , Neisseria/genética , Neisseria/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Escherichia coli has two DNA glycosylases for repair of DNA damage caused by simple alkylating agents. The inducible AlkA DNA glycosylase (3-methyladenine [m3A] DNA glycosylase II) removes several different alkylated bases including m3A and 3-methylguanine (m3G) from DNA, whereas the constitutively expressed Tag enzyme (m3A DNA glycosylase I) has appeared to be specific for excision of m3A. In this communication we have reexamined the substrate specificity of Tag by using synthetic DNA rich in GC base pairs to facilitate detection of any possible methyl-G removal. In such DNA alkylated with [3H]dimethyl sulphate, we found that m3G was excised from double-stranded DNA by both glycosylases, although more efficiently by AlkA than by Tag. This was further confirmed using both N-[3H]methyl-N-nitrosourea- and [3H]dimethyl sulphate-treated native DNA, from which Tag excised m3G with an efficiency that was about 70 times lower than for AlkA. These results can explain the previous observation that high levels of Tag expression will suppress the alkylation sensitivity of alkA mutant cells, further implying that m3G is formed in quantity sufficient to represent an important cytotoxic lesion if left unrepaired in cells exposed to alkylating agents.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Guanina/análogos & derivados , N-Glicosil Hidrolasas/metabolismo , Alquilación , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/metabolismo , Datos de Secuencia Molecular , Especificidad por Sustrato , Polimerasa TaqRESUMEN
An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.
Asunto(s)
ADN Glicosilasas , Escherichia coli/genética , Genes Bacterianos , Genes Fúngicos , N-Glicosil Hidrolasas/genética , Saccharomyces cerevisiae/genética , Adenina/análogos & derivados , Adenina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Reparación del ADN , Resistencia a Medicamentos , Expresión Génica , Guanina/análogos & derivados , Guanina/metabolismo , Metilmetanosulfonato/farmacología , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/biosíntesis , Sistemas de Lectura Abierta , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Ácido NucleicoRESUMEN
We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E. coli. These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15). Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts. Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression. The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure. Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression. These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA.
Asunto(s)
Adenina/metabolismo , Metanosulfonato de Etilo/toxicidad , Hipoxantina Fosforribosiltransferasa/genética , Metilmetanosulfonato/toxicidad , Mutación , N-Glicosil Hidrolasas/metabolismo , Adenina/análogos & derivados , Alquilación , Animales , Southern Blotting , Células CHO , Cricetinae , Cricetulus , ADN Glicosilasas , Escherichia coli/enzimología , Escherichia coli/genética , Hipoxantina Fosforribosiltransferasa/química , Cinética , N-Glicosil Hidrolasas/genéticaRESUMEN
The MAG gene of Saccharomyces cerevisiae encodes an alkylation repair DNA glycosylase whose sequence is homologous to the AlkA DNA glycosylase from Escherichia coli. To investigate the biochemical properties of MAG in comparison to AlkA, MAG was expressed in E. coli and purified to electrophoretic homogeneity. N-Terminal sequencing of the purified protein identified the translational start site which corresponded to that predicted previously from the nucleotide sequence. Polyclonal antibodies raised against MAG inhibited the enzymatic activity of MAG, but not that of AlkA, and vice versa, implying that the structures of the active site regions of these enzymes are antigenic, but sufficiently different to have different epitopes. Kinetic analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea and [3H]dimethyl sulfate showed that MAG was as effective as AlkA in removing 3-methyladenine, 7-methylguanine, and 7-methyladenine. However, the purified MAG enzyme did not catalyze the excision of O2-methylthymine, which is a major substrate for AlkA. Furthermore, 3-methylguanine was excised 20-40 times more slowly by MAG than by AlkA. The kinetics of 3-methylguanine excision by MAG were found to be similar to the low rate of 3-methylguanine excision catalyzed by 3-methyladenine DNA glycosylase I (Tag) of E. coli. Expression of MAG in alkA mutant cells did not effectively restore alkylation resistance of the mutant as did AlkA itself. It thus appears that MAG is a less versatile enzyme than AlkA in spite of the sequence relationship and may have a similar function in yeast as the nonhomologous Tag enzyme in E. coli.
Asunto(s)
Reparación del ADN , Genes Fúngicos , N-Glicosil Hidrolasas/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Alquilación , Secuencia de Aminoácidos , Clonación Molecular , ADN Glicosilasas , Escherichia coli/genética , Cinética , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Plásmidos , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Especificidad por Sustrato , Transformación GenéticaRESUMEN
One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.
Asunto(s)
Daño del ADN , Reparación del ADN , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , N-Glicosil Hidrolasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , ADN Glicosilasas , ADN de Hongos/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/metabolismo , Genes Fúngicos , Secuencias Hélice-Asa-Hélice , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/metabolismo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.
Asunto(s)
Ácido Apurínico/metabolismo , Reparación del ADN , Proteínas de Escherichia coli , Guanina/análogos & derivados , N-Glicosil Hidrolasas/genética , Pirimidinas/metabolismo , Secuencia de Aminoácidos , Mapeo Cromosómico , Clonación Molecular , ADN/metabolismo , ADN-Formamidopirimidina Glicosilasa , Escherichia coli/genética , Guanina/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutagénesis , N-Glicosil Hidrolasas/aislamiento & purificación , N-Glicosil Hidrolasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Especificidad por SustratoRESUMEN
The distributions of the mRNAs encoding the L-glutamate transporters GLT1 and GLAST were examined in the rat brain by in situ hybridization using 35S-labelled oligonucleotide probes. Probes directed to GLT1 produced dense labelling in the hippocampus, neocortex and neostriatum, and weak labelling in the cerebellum. In contrast, GLAST mRNA appeared to be greatly enriched in the cerebellum compared to other brain regions. While the intensity of the labelling for GLAST and GLT1 varied among different regions, their cellular distributions appeared to coincide inasmuch as both mRNAs were mainly expressed by glial cells. Labelling occurred, inter alia, in glial cells throughout the hippocampus, and in Golgi epithelial cells in the Purkinje cell layer of the cerebellum.