RESUMEN
Poly(ADP-ribose) polymerase 2 (PARP-2) is a DNA damage-dependent enzyme that belongs to a growing family of enzymes seemingly involved in genome protection. To gain insight into the physiological role of PARP-2 and to investigate mechanisms of PARP-2 gene regulation, we cloned and characterized the murine PARP-2 gene. The PARP-2 gene consists of 16 exons and 15 introns spanning about 13 kilobase pairs. Interestingly, the PARP-2 gene lies head to head with the gene encoding the mouse RNase P RNA subunit. The distance between the transcription start sites of the PARP-2 and RNase P RNA genes is 114 base pairs. This suggested that regulation of the expression of both genes may be coordinated through a bi-directional promoter. The PARP-2/RNase P RNA gene organization is conserved in the human. To our knowledge, this is the first report of a RNA polymerase II gene and an RNA polymerase III gene sharing the same promoter region and potentially the same transcriptional control elements. Reporter gene constructs showed that the 113-base pair intergenic region was indeed sufficient for the expression of both genes and revealed the importance of both the TATA and the DSE/Oct-1 expression control elements for the PARP-2 gene transcription. The expression of both genes is clearly independently regulated. PARP-2 is expressed only in certain tissues, and RNase P RNA is expressed in all tissues. This suggests that both genes may be subjected to multiple levels of control and may be regulated by different factors in different cellular contexts.
Asunto(s)
Endorribonucleasas/genética , Expresión Génica , Poli(ADP-Ribosa) Polimerasas/genética , Regiones Promotoras Genéticas/genética , ARN Catalítico/genética , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Genoma , Ratones , Datos de Secuencia Molecular , ARN/genética , Ribonucleasa PRESUMEN
Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.
Asunto(s)
ADN Ligasas/química , ADN Ligasas/metabolismo , Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Dedos de Zinc , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Huella de ADN , ADN Ligasa (ATP) , Desoxirribonucleasa I , Humanos , Lisina , Mamíferos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de XenopusRESUMEN
Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.
Asunto(s)
Daño del ADN/genética , Poli(ADP-Ribosa) Polimerasas/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Activación Enzimática/genética , Humanos , Hibridación Fluorescente in Situ , Linfocitos/enzimología , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
A dual approach to the study of poly (ADP-ribose)polymerase (PARP) in terms of its structure and function has been developed in our laboratory. Random mutagenesis of the DNA binding domain and catalytic domain of the human PARP, has allowed us to identify residues that are crucial for its enzymatic activity. In parallel PARP knock-out mice were generated by inactivation of both alleles by gene targeting. We showed that: (i) they are exquisitely sensitive to gamma-irradiation, (ii) they died rapidly from acute radiation toxicity to the small intestine, (iii) they displayed a high genomic instability to gamma-irradiation and MNU injection and, (iv) bone marrow cells rapidly underwent apoptosis following MNU treatment, demonstrating that PARP is a survival factor playing an essential and positive role during DNA damage recovery and survival.
Asunto(s)
Ratones Noqueados , Mutagénesis , Poli(ADP-Ribosa) Polimerasas/genética , Factores de Edad , Animales , Apoptosis , Peso Corporal , Catálisis , Escherichia coli/genética , Humanos , Ratones , Modelos Genéticos , Modelos Moleculares , Intercambio de Cromátides HermanasRESUMEN
Poly(ADP-ribose) polymerase (PARP) is a zinc-finger DNA binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these lesions, the immediate poly(ADP-ribosylation) of nuclear proteins converts DNA interruptions into intracellular signals that activate DNA repair or cell death programs. To elucidate the biological function of PARP in vivo, the mouse PARP gene was inactivated by homologous recombination to generate mice lacking a functional PARP gene. PARP knockout mice and the derived mouse embryonic fibroblasts (MEFs) were acutely sensitive to monofunctional alkylating agents and gamma-irradiation demonstrating that PARP is involved in recovery from DNA damage that triggers the base excision repair (BER) process. To address the issue of the role of PARP in BER, the ability of PARP-deficient mammalian cell extracts to repair a single abasic site present on a circular duplex plasmid molecule was tested in a standard in vitro repair assay. The results clearly demonstrate, for the first time, the involvement of PARP in the DNA synthesis step of the base excision repair process.
Asunto(s)
Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Daño del ADN , Células HeLa , Humanos , Ratones , Ratones Noqueados , Mutación , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase beta, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Células COS , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa beta/metabolismo , Escherichia coli , Células HeLa , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Saccharomyces cerevisiae , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Proteínas de Xenopus , Dedos de ZincRESUMEN
Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP-/- mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by gamma-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body gamma-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP-/- cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.
Asunto(s)
Daño del ADN , Poli(ADP-Ribosa) Polimerasas/fisiología , Alelos , Animales , Apoptosis/fisiología , Ciclo Celular/fisiología , Células Cultivadas , Femenino , Fibroblastos , Marcación de Gen , Ratones , Mutación , EmbarazoRESUMEN
Poly(ADP-ribose) polymerase (PARP) has been suggested to play a regulatory role in vivo, in DNA replication and/or DNA repair based mainly on its capacity to bind to DNA strand breaks. This interaction is modulated through auto poly(ADP-ribosylation). However, the biological function of PARP may also involve interactions with proteins such as topoisomerase I or DNA polymerase alpha, which may or may not be themselves ADP-ribosylated. Using the yeast two-hybrid method search for other proteins interacting with PARP, we have isolated a full-length cDNA clone coding for a protein of 158 amino acid residues. This amino acid sequence is 66 and 56% identical to yeast ubiquitin-conjugating enzymes Hus5 and Ubc9 of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Moreover, we have demonstrated that the expressed protein complements a S. cerevisiae yeast strain deficient for Ubc9. The protein encoded by the isolated cDNA is thus a new human counterpart of the ubiquitin-conjugating enzyme family and has been called hUbc9. The hubc9 gene locus has been assigned to the chromosomal location 16p13.2-p13.3. By means of two-hybrid analysis it was discovered that hUbc9 interacts with the automodification domain of PARP. This interaction was further confirmed using GST (glutathione-S-transferase) tagged fusion proteins: (i) in vivo, by transfecting cos7 cells with hUbc9 cloned in an eukaryotic expression vector, and (ii) in vitro, by mixing purified PARP with hUbc9 purified and expressed in bacteria. The possible significance and function of this interaction is discussed while taking into account the possible intracellular role of hUbc9.
Asunto(s)
Ligasas/genética , Ligasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Enzimas Ubiquitina-Conjugadoras , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Western Blotting , Células COS , Ciclo Celular/genética , Mapeo Cromosómico , Cromosomas Humanos Par 16 , ADN Complementario/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Biblioteca de Genes , Prueba de Complementación Genética , Células HeLa , Humanos , Células Híbridas , Datos de Secuencia Molecular , Mutación , Poli(ADP-Ribosa) Polimerasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Levaduras/genéticaRESUMEN
Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.
Asunto(s)
Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Catálisis , Pollos , Cristalización , Cristalografía por Rayos X , Daño del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Genoma Humano , Células HeLa , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Relación Estructura-Actividad , TransfecciónRESUMEN
Poly (ADP-ribose) polymerase (PARP) participates in the immediate response in mammalian cells exposed to DNA-damaging agents. Recombinant baculovirus harboring the cDNA of the chicken PARP catalytic domain (40 kDa) have been used to infect Spodoptera frugiperda (Sf9) insect cells. The recombinant polypeptide (30 mg per 1 x 10(9) cells) was purified to homogeneity by 3-aminobenzamide affinity chromatography. The enzymatic properties of the recombinant domain were similar to those of the native fragment. Crystals of the purified recombinant catalytic domain were grown by vapor diffusion. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 59.2 A, b = 65.0 A, c = 96.9 A. They are suitable for X-ray analysis and diffract to 2.0 A.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/química , Animales , Baculoviridae/genética , Secuencia de Bases , Sitios de Unión , Pollos , Cromatografía de Afinidad , Cristalografía por Rayos X , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Spodoptera/citologíaRESUMEN
Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genetoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and the function of this abundant and highly conserved enzyme. This article summarizes our present knowledge in this field.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/química , Poli(ADP-Ribosa) Polimerasas/fisiología , Secuencia de Aminoácidos , Animales , Catálisis , ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Poly(ADPR)polymerase (poly(ADPR)P) mRNA and enzymatic activity levels were investigated in primary cultures of rat astrocytes and neurons in the absence or presence of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), respectively. In cultured rat astrocytes, a biphasic increase in poly(ADPR)P mRNA, associated with enhanced nuclear poly(ADPR)P enzymatic activity, were observed. The first rise in poly(ADPR)P mRNA and enzymatic activity is at the beginning of cell proliferation and the second with the occurrence of cell differentiation. In the presence of bFGF (5 ng/ml) the mRNA peaks and the differentiation-associated poly(ADPR)P enzymatic activity undergoes a 2-fold increase. In neuronal cultures an initial high level of poly(ADPR)P mRNA is followed by a decrease while differentiation is progressively achieved. A limited increase of poly(ADPR)P activity is observed during this phase. In the presence of NGF (50 ng/ml), similar poly(ADPR)P mRNA expression and enzymatic activity patterns are observed. The results suggest that poly(ADPR)P is involved at the onset of nerve-cell proliferation and differentiation.
Asunto(s)
Astrocitos/citología , Neuronas/citología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Astrocitos/enzimología , Northern Blotting , Diferenciación Celular/genética , División Celular/genética , Células Cultivadas , Factores de Crecimiento de Fibroblastos/fisiología , Regulación Enzimológica de la Expresión Génica , Cinética , Neuronas/enzimología , Poli(ADP-Ribosa) Polimerasas/genética , Ratas , Ratas EndogámicasRESUMEN
The complete nucleotide (nt) sequence of the cDNA encoding the chicken poly(ADP-ribose) synthetase has been determined. Positive clones overlapping the 5' region or the 3' region of the cDNA have been isolated from a lambda gt 10 hen oviduct cDNA library using two human cDNA probes. The missing middle portion has been obtained by the polymerase chain reaction procedure. A single 3033-nt open reading frame from start codon to stop codon encodes a sequence of 1011 amino acid residues. The alignment of this sequence with those from human and mouse reveals overall identities of 79% and 77%, respectively. However, an identity of about 82% is obtained in the DNA-binding domain within the two zinc fingers, and an even higher similarity (85-87%) is observed in the NAD-binding domain. The isolated clones consistently hybridize on chicken Northern blots to an mRNA species of about 4 kb, whereas they do not cross-hybridize with RNA blots of Drosophila melanogaster. Thus, it appears that, even if the functional properties of the enzyme are maintained, the cDNA identity will be much decreased in nonvertebrate organisms.
Asunto(s)
Poli(ADP-Ribosa) Polimerasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Pollos , Clonación Molecular , ADN , Humanos , Mamíferos/genética , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
A homogeneous preparation of an arginine-specific mono(ADP-ribosyl)transferase from turkey erythrocytes effectively utilized 2'-deoxy-NAD+ for the 2'-deoxy(ADP-ribose) modification of arginine methyl ester with an apparent Km of 27.2 microM and a Vmax of 36.4 mumol min-1 (mg of protein)-1. The adduct formed was also used as a substrate by an avian erythrocyte arginine(ADP-ribose)-specific hydrolase that generated free 2'-deoxy(ADP-ribose). In contrast, 2'-deoxy-NAD+ was not a substrate in the initiation or elongation reaction catalyzed by highly purified poly(ADP-ribose) polymerase from calf thymus. However, 2'-deoxy-NAD+ was a potent noncompetitive inhibitor of NAD+ in the elongation reaction catalyzed by the polymerase, with an apparent Ki of 32 microM. These results indicate that 2'-deoxy-NAD+ may be utilized to specifically identify protein acceptors for endogenous mono(ADP-ribosyl)transferases in complex biological systems that may contain a high activity of poly(ADP-ribose) polymerase, i.e., cell nuclei preparations.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , NAD/análogos & derivados , ADP Ribosa Transferasas , Animales , Bovinos , Eritrocitos/enzimología , Cinética , NAD/metabolismo , Oxidación-Reducción , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
Poly(ADP-ribose) polymerase associated with free cytoplasmic messenger ribonucleoprotein particles (mRNP) has been characterized in mouse plasmacytoma. This cytoplasmic enzyme undergoes auto-ADP-ribosylation and has a similar molecular weight and common antigenic sites with the chromatin bound poly(ADP-ribose) polymerase in spite of its DNA independency. The free mRNP poly(ADP-ribose) polymerase is released from the particle only by high saline concentrations (0.7 M KCl) and the dissociated enzyme expresses a higher activity. The treatment of free mRNP by RNase A stimulates the poly(ADP-ribose) polymerase activity. Partial destruction of mRNP by high saline concentration or mRNA digestion unmasks new protein sites for ADP-ribosylation. In view of the changes that occur in the free mRNP structure to permit mRNA translation, a possible role of poly(ADP-ribosylation) as an important post-synthetic modification of some of the mRNP proteins is discussed.
Asunto(s)
Citoplasma/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Autorradiografía , Citoplasma/enzimología , ADN/fisiología , Desoxirribonucleasa I/metabolismo , Ratones , Ratones Endogámicos BALB C/metabolismo , Peso Molecular , Proteínas de Neoplasias/metabolismo , Plasmacitoma/enzimología , Plasmacitoma/metabolismo , Cloruro de Potasio/farmacología , Biosíntesis de Proteínas , Ribonucleasa Pancreática/metabolismoRESUMEN
The pattern of nucleosomal histones poly(ADP-ribosyl)ation is changed under conditions which affect the poly(ADP-ribosyl)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP-ribosyl)ate histones H1 and H1, H2A, A2A, and H2B. However at NAD concentrations above 10 microM the enzyme preferentially poly(ADP-ribosyl)ates histone H1 to a hyper ADP-ribosylated form. Furthermore we have observed hyper ADP-ribosylation of histone H2B at NAD concentrations of 10 microM suggesting that histone H2B can undergo the same type of ADP-ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.
Asunto(s)
Histonas/metabolismo , NAD+ Nucleosidasa/metabolismo , NAD/farmacología , Nucleosomas/enzimología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Animales , Sitios de Unión , Bovinos , Cromatina/metabolismo , Reparación del ADN , Replicación del ADN , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Timo/enzimologíaRESUMEN
Isolated rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADP-ribose) polymerase. A time course study was performed using an NAD concentration of 200 microM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP-ribosylation) and determinations of DNA polymerase alpha activity on ADP-ribosylated polynucleosomes were done at different time intervals. A direct kinetic correlation between ADP-ribose incorporation, polynucleosome relaxation amd histone H1 hyper-ADP-ribosylation was established. In addition, DNA polymerase alpha activity was highly stimulated on ADP-ribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action. Because of the strong evidence implicating histone H1 in the maintenance of higher-ordered chromatin structures, the present study may provide a basis for the interpretation of the involvement of the histone H1 ADP-ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.
Asunto(s)
Adenosina Difosfato Ribosa/metabolismo , Histonas/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , Nucleosomas/metabolismo , Animales , Bovinos , ADN Polimerasa II/metabolismo , Cinética , Microscopía Electrónica , Páncreas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Ratas , UltracentrifugaciónRESUMEN
ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP. This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis. Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.
Asunto(s)
Nucleotidiltransferasas/metabolismo , Plasmacitoma/enzimología , Ribonucleoproteínas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Animales , Citoplasma/metabolismo , Hidrolasas/farmacología , Ratones , Nucleotidiltransferasas/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasas , Ribonucleasa Pancreática/farmacologíaRESUMEN
A slight DNA topoisomerase I activity was detected in highly purified poly(ADP-Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP-ribosylation) reaction occurs in the presence of NAD. Purified topoisomerase I from calf thymus was shown to be ADP-ribosylated by poly(ADP-Rib) polymerase purified from the same tissue. Poly(ADP-ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP-ribosylation. The fact that a slight poly(ADP-Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that topoisomerase I activity can be modified by ADP-ribosylation, may suggest a spatial and functional correlation of these two enzymes in chromatin.
Asunto(s)
ADN-Topoisomerasas de Tipo I/aislamiento & purificación , NAD+ Nucleosidasa/aislamiento & purificación , Poli(ADP-Ribosa) Polimerasas/aislamiento & purificación , Timo/enzimología , Animales , Bovinos , Fenómenos Químicos , Química , Técnicas In Vitro , Cinética , Poli Adenosina Difosfato Ribosa/farmacología , Unión Proteica , Inhibidores de Topoisomerasa IRESUMEN
Interaction of calf thymus poly(ADP-ribose(ADPR] polymerase with a copurified DNA fraction (sDNA) was investigated. Electron microscopic studies of sDNA which appeared to be a powerful poly(ADPR) polymerase activator have shown that the purified poly(ADPR) polymerase-DNA complexes possess a "nucleosome like structure", with DNA wrapping around the enzyme molecule. Examination of the DNA linked poly(ADPR) polymerase preparations revealed the presence of Y-structures in sDNA. The enrichment in the sDNA fraction of the Y shape DNA suggests the existence of replication fork structures in the poly(ADPR) polymerase linked DNA and or in the vicinity of the enzyme. With increasing auto-poly(ADP-ribosyl)ation the enzyme molecule becomes much denser, increases in size and detaches from the DNA. When poly(ADPR) formed was purified and examined by electron microscopy, branched polymers of different sizes were observed. The formation of these polymers may explain the size gained by poly ADP-ribosylated enzyme molecules. When the interaction of poly(ADPR) polymerase with the plasmid pBR 322 was tested, a slight contamination of our enzyme preparation with topoisomerase I was detected. The contaminant topoisomerase I activity, however, was completely abolished by ADP-ribosylation. Further experiments with purified calf thymus topoisomerase I confirmed that this enzyme loses its activity following ADP-ribosylation with poly(ADPR) polymerase. These results may suggest that ADP-ribosylation of topoisomerase I can be one of the regulatory mechanisms of its activity. Furthermore, these results confirm that a topoisomerase I contaminant does not interfere with the ADP-ribosylation experiments of purified poly(ADPR) polymerase preparation.