RESUMEN
New functions have been identified for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds specifically to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the beta-amyloid precursor protein and the huntingtin protein. However, the pathophysiological significance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole-cell sonicates of Alzheimer's and Huntington's disease fibroblasts. However, subcellular-specific GAPDH-protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole-cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH-protein distribution as a function of its subcellular localization in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age-matched controls. In the nuclear fraction, deficits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH:neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Fibroblastos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/deficiencia , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Enfermedad de Huntington/enzimología , Anciano , Enfermedad de Alzheimer/patología , Células Cultivadas , Activación Enzimática , Femenino , Fibroblastos/patología , Humanos , Enfermedad de Huntington/patología , Immunoblotting , L-Lactato Deshidrogenasa/metabolismo , Masculino , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional , Fracciones SubcelularesRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Animales , Apoptosis , Reparación del ADN , Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Masculino , Fusión de Membrana , Enfermedades Neurodegenerativas/enzimología , Óxido Nítrico/química , Fosforilación , Neoplasias de la Próstata/enzimología , Relación Estructura-Actividad , Tubulina (Proteína)/químicaRESUMEN
Pea (Pisum sativum) chloroplastic glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) was tested for uracil DNA glycosylase activity. It was found that both the chloroplast and the recombinant subunit B dehydrogenases remove uracil from poly(dA[3H]dU). The glycosylase activity of the recombinant subunit B enzyme and that of a truncated form corresponding in length to subunit A were associated with the dehydrogenase activity in gel-filtration experiments. Both activities of the chloroplast enzyme were inhibited by antisera raised against recombinant subunit B, and both activities of the recombinant subunit B enzyme were inhibited by antisera raised against pea chloroplast glyceraldehyde-3-P dehydrogenase. Antisera raised against Escherichia coli uracil glycosylase did not affect the glycosylase activity of the recombinant subunit B enzyme. The glycosylase pH activity profile of the chloroplast dehydrogenase was unique. It is distinct from the dehydrogenase pH activity profile and from the pH activity profiles of other plant glycosylases. The glycosylase activity, but not the dehydrogenase activity, of the recombinant subunit B enzyme was inhibited by uracil. Pyridine nucleotides stimulated the glycosylase activity. To our knowledge this is the first example of a nonhuman glyceraldehyde-3-P dehydrogenase, and of an NADP-dependent glyceraldehyde-3-P dehydrogenase, that exhibits uracil glycosylase activity.
Asunto(s)
Cloroplastos/enzimología , ADN Glicosilasas , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , Pisum sativum/enzimología , Secuencia de Aminoácidos , Cromatografía en Gel , Relación Dosis-Respuesta a Droga , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , N-Glicosil Hidrolasas/inmunología , Factores de Tiempo , Uracilo/metabolismo , Uracil-ADN GlicosidasaRESUMEN
The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested.
Asunto(s)
Encefalopatías/enzimología , Fenómenos Fisiológicos Celulares , Células/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/fisiología , Animales , Encefalopatías/patología , Células/patología , Glucólisis , HumanosRESUMEN
Recent evidence indicates new, intriguing roles for the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in fundamental mammalian cell processes. These include its role in DNA repair, in the translational control of gene expression, in DNA replication and in endocytosis. These findings have the potential to alter our basic understanding of the molecular mechanisms through which human or mammalian cells utilize individual proteins in vital, yet unrelated, cell processes.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/fisiología , Animales , Reparación del ADN , Replicación del ADN , Endocitosis , Regulación Enzimológica de la Expresión Génica , Glucólisis , Humanos , Biosíntesis de ProteínasRESUMEN
5-Fluorouracil (5-FlUra), a cancer chemotherapeutic agent used in the treatment of colon, breast, ovarian and prostate cancer, is incorporated into DNA as a result of its utilization as 5-FldUTP during DNA synthesis. This promutagenic DNA lesion is excised by the base excision repair enzyme uracil DNA glycosylase (UDG). In this report we describe for the first time a mechanism by which 5-FlUra as the free base specifically binds in vivo to the UDG in noncycling human cells, thereby inhibiting its activity. By using 5-FlUra concentrations which did not elicit demonstrable cell toxicity, a dose-dependent decrease in UDG activity was detected which approached 30% of that observed in control cells. In contrast, exposure of cells to equivalent concentrations of uracil, 5-fluorodeoxyuridine or 5-bromouracil had no effect on UDG activity. Subsequent studies demonstrated a reversible binding of 5-FlUra to the glycosylase. Kinetic analysis using nonlinear regression analysis demonstrated a competitive mode of inhibition and indicated a tight binding of 5-FlUra to UDG in vivo, although the 5-FlUra-UDG complex was easily dissociated in vitro. These findings describe a potentially new and novel mechanism of action of 5-FlUra in a nonproliferating human cell population. The potential relevance of these findings to the utility of 5-FlUra as a cancer chemotherapeutic agent is considered.
Asunto(s)
ADN Glicosilasas , Fluorouracilo/farmacología , N-Glicosil Hidrolasas/antagonistas & inhibidores , Unión Competitiva , Recuento de Células/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Floxuridina/farmacología , Humanos , Cinética , Uracilo/farmacología , Uracil-ADN GlicosidasaRESUMEN
Recent evidence indicates that 5-fluorouracil (5-FlUra) is incorporated into DNA and is removed by the DNA repair enzyme uracil DNA glycosylase. Synthetic oligonucleotides containing either a single uracil or 5-FlUra residue were constructed to examine the mechanisms by which human cells remove 5-FlUra from DNA. The human uracil DNA glycosylase excised uracil in a manner similar to that observed for the bacterial enzyme. In contrast, a significant difference was observed in their abilities to remove 5-FlUra. In particular, both the bacterial and normal human enzymes displayed 13-17-fold increases in their apparent Km values but the apparent Vmax values remained virtually constant. These results demonstrate that normal human cells possess a defined capacity to remove 5-FlUra incorporated into DNA. However, specific kinetic differences may exist that affect their capacity to remove 5-FlUra formed in DNA after treatment with this cancer chemotherapeutic agent.
Asunto(s)
ADN Glicosilasas , ADN/metabolismo , Fluorouracilo/metabolismo , N-Glicosil Hidrolasas/metabolismo , Secuencia de Bases , ADN/química , Reparación del ADN , Escherichia coli/enzimología , Humanos , Cinética , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Uracil-ADN GlicosidasaRESUMEN
The cell cycle regulation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene was examined in normal human cells. Steady state RNA levels were monitored by Northern blot analysis using a plasmid (pChug 20.1) which contained the 1.3 kb GAPDH/UDG cDNA. The biosynthesis of the 37 kDa GAPDH/UDG protein was determined using an anti-human placental GAPDH/UDG monoclonal antibody to immunoprecipitate the radiolabeled protein. Increases in steady state GAPDH/UDG mRNA levels were cell cycle specific. A biphasic pattern was observed resulting in a 19-fold increase in the amount of GAPDH/UDG mRNA. The biosynthesis of the 37 kDa GAPDH/UDG protein displayed a similar biphasic regulation with a 7-fold increase. Pulse-chase experiments revealed a remarkably short half life of less than 1 hr. for the newly synthesized 37 kDa protein, comparable to that previously documented for a number of oncogenes. GAPDH/UDG mRNA levels were markedly reduced at 24 hr. when DNA synthesis was maximal. These results define the GAPDH/UDG gene as cell cycle regulated with a characteristic temporal sequence of expression in relation to DNA synthesis. The cell cycle synthesis of a labile 37 kDa monomer suggests a possible regulatory function for this multidimensional protein. Further, modulation of the GAPDH/UDG gene in the cell cycle may preclude its use as a reporter gene when the proliferative state of the cell is not kept constant.
Asunto(s)
Ciclo Celular/genética , ADN Glicosilasas , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , N-Glicosil Hidrolasas/genética , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , N-Glicosil Hidrolasas/biosíntesis , ARN Mensajero/metabolismo , Fase S , Uracil-ADN GlicosidasaRESUMEN
The developmental regulation of the mammalian DNA-repair enzyme uracil DNA glycosylase was examined in the rat at specific intervals ranging from -4 days before to 106 days after birth. Enzyme activity was quantitated by in vitro biochemical assay. In the adult animal, as measured in crude cell extracts, three organs (liver, kidney and spleen) had significant levels of activity. In contrast, three organs (brain, heart and lung) had low activity. Partial purification of this enzyme identified one major species of molecular weight 32,700 Da, demonstrating the quantitation of the nuclear glycosylase. During development, with the exception of the liver, the specific activity of the glycosylase paralleled the regulation of DNA synthesis. In these organs the highest levels of the glycosylase and the rate of DNA replication were observed around the time of birth. In the liver, DNA replication was similarly regulated. However, glycosylase activity was minimal at early stages of life. Instead, maximal levels were observed at 14-21 days after birth. At that time DNA replication was severely reduced. These results demonstrate that individual organs express this DNA-repair enzyme in a distinct and specific pattern during development. Accordingly, the regulation of the uracil DNA glycosylase during development may provide a model system to examine the differential regulation of DNA-repair genes.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , N-Glicosil Hidrolasas/metabolismo , Factores de Edad , Animales , División Celular , Replicación del ADN , Femenino , Ratas , Ratas Sprague-Dawley/embriología , Distribución Tisular , Uracilo/metabolismo , Uracil-ADN GlicosidasaRESUMEN
The relationship between the proliferative dependent expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)/uracil DNA glycosylase (UDG) gene and the induction of uracil DNA glycosylase activity was examined in human cells. Three different cell types were studied to determine whether the growth-dependent regulation of this multifunctional gene was a common characteristic of human cells. These included WI-38 normal embryonic lung fibroblasts, a Japanese Bloom's syndrome non-transformed skin fibroblast cell strain (GM-05289) and a lymphoblastoid cell line transformed by the Epstein-Barr virus. The Japanese Bloom's syndrome cells displayed the altered immunoreactivity with marker monoclonal antibody 40.10.09 which characterizes cells from this human genetic disorder. In noncycling human cells Northern blot analysis using a plasmid (pChug 20.1) which contained the human GAPDH/UDG cDNA revealed a single 1.6 kb transcript. In each case, the expression of this gene was increased during cell proliferation. This increase in GAPDH/UDG gene expression was identical to that observed for UDG enzyme activity. Further, using anti-human UDG monoclonal antibodies, there was a growth-dependent rise in immunoreactivity suggesting an increase in the level of antigenic protein. These results demonstrate that: (i) the expression of the GAPDH/UDG gene was dependent on the proliferative state of the cell; and (ii) a correlation existed between the transcription of this gene and the level of uracil DNA glycosylase enzyme activity.
Asunto(s)
División Celular , ADN Glicosilasas , Reparación del ADN , Regulación Enzimológica de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , N-Glicosil Hidrolasas/genética , Northern Blotting , Transformación Celular Viral , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , N-Glicosil Hidrolasas/metabolismo , Transcripción Genética , Uracil-ADN GlicosidasaRESUMEN
Bloom's syndrome uracil DNA glycosylase was highly purified from two non-transformed cell strains derived from individuals from different ethnic groups. Their properties were then compared to two different highly purified normal human uracil DNA glycosylases. A molecular mass of 37 kDa was observed for each of the four human enzymes as defined by gel-filtration column chromatography and by SDS-PAGE. Each of the 37 kDa proteins was identified as a uracil DNA glycosylase by electroelution from the SDS polyacrylamide gel, determination of glycosylase activity by in vitro biochemical assay and identification of the reaction product as free uracil by co-chromatography with authentic uracil. Bloom's syndrome enzymes differed substantially in their isoelectric point and were thermolabile as compared to the normal human enzymes. Bloom's syndrome enzymes displayed a different Km, Vmax and were strikingly insensitive to 5-fluorouracil and 5-bromouracil, pyrimidine analogues which drastically decreased the activity of the normal human enzymes. In particular, each Bloom's syndrome enzyme required 10-100-fold higher concentrations of each analogue to achieve comparable inhibition of enzyme activity. Potential mechanisms are considered through which an altered uracil DNA glycosylase characterizing this cancer-prone human genetic disorder may arise.
Asunto(s)
Síndrome de Bloom/enzimología , ADN Glicosilasas , Isoenzimas , Judíos/genética , N-Glicosil Hidrolasas/química , Población Negra/genética , Síndrome de Bloom/etnología , Estabilidad de Enzimas , Fibroblastos/química , Humanos , Punto Isoeléctrico , Cinética , N-Glicosil Hidrolasas/aislamiento & purificación , Polinucleótidos/metabolismo , Timina/metabolismo , Estados Unidos/etnología , Uracilo/análogos & derivados , Uracilo/metabolismo , Uracil-ADN GlicosidasaRESUMEN
We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.
Asunto(s)
ADN Glicosilasas , Gliceraldehído-3-Fosfato Deshidrogenasas/química , N-Glicosil Hidrolasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Síndrome de Bloom/enzimología , Síndrome de Bloom/genética , Western Blotting , Núcleo Celular/enzimología , Clonación Molecular , ADN/genética , Reparación del ADN , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Uracil-ADN GlicosidasaRESUMEN
The regulation of the base excision repair enzymes uracil DNA glycosylase and hypoxanthine DNA glycosylase was examined in 2 different progeroid cell strains. The immunoreactivity of the uracil DNA glycosylase in progeroid cells was examined by enzyme linked immunosorbent assay (ELISA) and by immunoblot analysis. The enzyme was recognized in a quantitative manner by 2 different anti-human uracil DNA glycosylase monoclonal antibodies in the ELISA. Western blot analysis identified a glycosylase protein of Mr = 37,000. In randomly proliferating progeroid cells, the uracil DNA glycosylase was enhanced 3-fold during cell growth. In synchronous cells, uracil DNA glycosylase and hypoxanthine DNA glycosylase were induced with an extent of induction (5-6-fold) comparable to that observed for normal human cells. Further, the activity of each base excision repair enzyme was enhanced with a comparable temporal sequence prior to the induction of DNA synthesis and DNA polymerase activity. These results indicate a normal cell cycle regulation of base excision repair in progeroid cells.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , Glicósido Hidrolasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , Progeria/enzimología , Anticuerpos Monoclonales , Ciclo Celular , División Celular , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/enzimología , Glicósido Hidrolasas/análisis , Homeostasis , Humanos , Peso Molecular , N-Glicosil Hidrolasas/análisis , Progeria/genética , Valores de Referencia , Uracil-ADN GlicosidasaRESUMEN
The immunological reactivity of the uracil DNA glycosylase was investigated in three Epstein-Barr virus-transformed human lymphoblastoid cell lines. Two were derived from normal human lymphocytes while the third was derived from a Bloom's syndrome patient. A panel of 3 anti-human placental uracil DNA glycosylase monoclonal antibodies (37.04.12, 40.10.09 and 42.08.07) was used. Immunological reactivity was determined in a double-blind enzyme-linked immunosorbent assay (ELISA); by inhibition of enzyme activity; and by immunoblot analysis. In the ELISA, the glycosylase from each lymphoblastoid cell line was recognized by glycosylase antibodies 37.04.12 and 42.08.07. In contrast, antibody 40.10.09 failed to recognize the glycosylase from the Bloom's syndrome cell line. Further analysis demonstrated that the 40.10.09 antibody was unable to inhibit catalysis by the Bloom's syndrome lymphoblast glycosylase. In contrast, the 40.10.09 antibody inhibited the activity of the two normal human lymphoblast enzymes. Denaturation of the Bloom's syndrome lymphoblast glycosylase rendered that protein immunoreactive with the 40.10.09 antibody. These results demonstrated that: (1) the immunological alteration in the Bloom's syndrome uracil DNA glycosylase was detected in hematopoietic cells; and (2) viral transformation did not affect the immunoreactivity of the enzyme from either normal human or Bloom's syndrome cells.
Asunto(s)
Síndrome de Bloom/inmunología , ADN Glicosilasas , N-Glicosil Hidrolasas/inmunología , Anticuerpos Monoclonales , Síndrome de Bloom/enzimología , Síndrome de Bloom/genética , Línea Celular Transformada , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/enzimología , Herpesvirus Humano 4 , Humanos , Immunoblotting , N-Glicosil Hidrolasas/antagonistas & inhibidores , N-Glicosil Hidrolasas/metabolismo , Uracil-ADN GlicosidasaRESUMEN
This critical review considers recent work on alterations in DNA repair capacity in Bloom's syndrome as a molecular mechanism for this human disorder. Four main types of DNA repair deficiencies are discussed. These include perturbations in the temporal regulation of DNA repair pathways during the cell cycle, failure to enhance DNA repair pathways during cell proliferation, reduced levels of DNA ligase in Bloom's syndrome cells, and the identification of mutant repair enzyme proteins. These deficiencies are considered in relation to the cellular characteristics of Bloom's syndrome, including delays in DNA replication, hypermutability, and increased incidence of chromosomal aberrations (spontaneously occurring or observed after exposure to environmental agents). The relationship between DNA repair deficiencies and the genetic basis of Bloom's syndrome is described. Previous evidence suggested an autosomal recessive mode of inheritance for Bloom's syndrome. A discussion is presented as to the molecular mechanism through which an alteration in a single gene could result in multiple DNA repair defects.
Asunto(s)
Síndrome de Bloom/genética , ADN Glicosilasas , Reparación del ADN , N-Glicosil Hidrolasas/metabolismo , Secuencia de Bases , Línea Celular , ADN/genética , Replicación del ADN , Humanos , Datos de Secuencia Molecular , Uracil-ADN GlicosidasaRESUMEN
A series of anti-human placental uracil DNA glycosylase monoclonal antibodies was used to screen a human placental cDNA library in phage lambda gt11. Twenty-seven immunopositive plaques were detected and purified. One clone containing a 1.2-kilobase (kb) human cDNA insert was chosen for further study by insertion into pUC8. The resultant recombinant plasmid selected by hybridization a human placental mRNA that encoded a 37-kDa polypeptide. This protein was immunoprecipitated specifically by an anti-human placental uracil DNA glycosylase monoclonal antibody. RNA blot-hybridization (Northern) analysis using placental poly(A)+ RNA or total RNA from four different human fibroblast cell strains revealed a single 1.6-kb transcript. Genomic blots using DNA from each cell strain digested with either EcoRI or Pst I revealed a complex pattern of cDNA-hybridizing restriction fragments. The genomic analysis for each enzyme was highly similar in all four human cell strains. In contrast, a single band was observed when genomic analysis was performed with the identical DNA digests with an actin gene probe. During cell proliferation there was an increase in the level of glycosylase mRNA that paralleled the increase in uracil DNA glycosylase enzyme activity. The isolation of the human uracil DNA glycosylase gene permits an examination of the structure, organization, and expression of a human DNA repair gene.
Asunto(s)
ADN Glicosilasas , ADN/aislamiento & purificación , Genes , N-Glicosil Hidrolasas/genética , División Celular , Línea Celular , ADN/genética , Femenino , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genoma Humano , Humanos , Cinética , Placenta/enzimología , Embarazo , Transcripción Genética , Uracil-ADN GlicosidasaRESUMEN
The physical association of mammalian excision repair enzymes with DNA was examined as a function of cell proliferation. The molecular weight distribution of two nuclear base excision repair enzymes, the uracil DNA glycosylase and the hypoxanthine DNA glycosylase, were examined by sucrose step gradient analysis. The sedimentation of DNA polymerase activity as well as the distribution of parental and replicating DNA were determined simultaneously. In confluent BHK-21 fibroblasts, basal levels of both DNA glycosylases, DNA polymerase beta, and parental DNA sedimented to the 20%/40% sucrose border. In proliferating BHK-21 cells, induced levels of both DNA glycosylases, DNA polymerase alpha, and replicating DNA sedimented to the 40%/50% sucrose border. The physical association of the repair enzymes with DNA was demonstrated by detergent treatment and by DNase digestion. As defined by [35S] methionine pulse labeling analysis, newly synthesized DNA repair enzymes were localized with either parental or replicating DNA. These results suggested that the physical association of mammalian DNA repair enzymes with nuclear DNA was dependent on the proliferative state of the cell.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , Glicósido Hidrolasas/metabolismo , N-Glicosil Hidrolasas/metabolismo , División Celular , Células Cultivadas , Centrifugación por Gradiente de Densidad , Humanos , Peso Molecular , Uracil-ADN GlicosidasaRESUMEN
The immunocytochemical localization of the base excision repair enzyme uracil DNA glycosylase was examined as a function of cell proliferation. Two nontransformed normal human fibroblast cell strains were analyzed using an anti-human uracil DNA glycosylase monoclonal antibody. In quiescent cells, basal levels of a nonnuclear immunocytochemically reactive glycosylase protein were detected. No nuclear immunofluorescence was observed. In contrast, in proliferating cells, intense immunofluorescence could be detected exclusively in the nuclear or perinuclear regions. As proliferation diminished, basal levels of the nonnuclear immunocytochemically reactive glycosylase were again observed. The subcellular distribution of the glycosylase was examined in parallel by in vitro biochemical assay. In quiescent cells, glycosylase activity was observed in both the nuclear and membrane fractions. A small amount of enzyme activity could be detected in the soluble cytoplasmic fraction. Immunoblot analysis demonstrated a Mr 37,000 glycosylase protein in each subcellular fraction. During cell proliferation, there was an increase in glycosylase activity in each of the subcellular fractions. These results suggest a correlation between the proliferative state of normal human cells and the preferential nuclear or perinuclear localization of an immunocytochemically reactive glycosylase protein. Further, immunofluorescence of the nuclear enzyme may be dependent on defined conformational states of that nuclear glycosylase in the cell cycle.
Asunto(s)
Anticuerpos Antinucleares , División Celular , Núcleo Celular/enzimología , ADN Glicosilasas , Reparación del ADN , ADN/biosíntesis , N-Glicosil Hidrolasas/análisis , Células Cultivadas , Humanos , Uracil-ADN GlicosidasaRESUMEN
Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Síndrome de Bloom/enzimología , ADN Glicosilasas , N-Glicosil Hidrolasas/inmunología , Síndrome de Bloom/diagnóstico , Síndrome de Bloom/inmunología , Línea Celular , Reparación del ADN , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/enzimología , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/enzimología , Humanos , N-Glicosil Hidrolasas/genética , Uracil-ADN GlicosidasaRESUMEN
The immunoreactivity of normal human and Bloom's syndrome uracil DNA glycosylase was examined using a series of three anti-human placental uracil DNA glycosylase monoclonal antibodies. Immunoreactivity was determined by three separate and independent criteria: enzyme-linked immunosorbent assay (ELISA), enzyme inhibition studies and immunoblot analysis. As defined by each criteria, normal human uracil DNA glycosylase was immunoreactive with each antibody (37.04.12, 40.10.09 and 42.08.07). In contrast, each glycosylase purified from two separate non-transformed Bloom's syndrome cell strains was not reactive with antibody 40.10.09. First, no ELISA reactivity was observed with each glycosylase protein. Second, catalysis by each Bloom's syndrome glycosylase was not inhibited by antibody 40.10.09. However, each Bloom's syndrome enzyme was immunoreactive with antibodies 37.04.12 and 42.08.07. No immunoreactive glycosylase species was observed during the induction of the Bloom's syndrome enzyme during cell proliferation. However, immunoreactivity of the denatured Bloom's syndrome enzyme with 40.10.09 antibody was observed by immunoblot analysis. These results suggest that Bloom's syndrome uracil DNA glycosylase is characterized by a structural alteration in the native glycosylase protein secondary to the primary antigenic site recognized by the 40.10.09 antibody. This altered antigenicity may provide an immunological marker for the identification of this human genetic syndrome.