RESUMEN
H1 RNA, the RNA component of the human nuclear RNase P, is encoded by a unique gene transcribed by RNA polymerase III (Pol III). In this work, cis-acting elements and trans-acting factors involved in human H1 gene transcription were characterized by transcription assays of mutant templates and DNA binding assays of recombinant proteins. Four elements, lying within 100 bp of 5'-flanking sequences, were defined to be essential for maximal in vitro and in vivo expression, consisting of the octamer, Staf, proximal sequence element (PSE) and TATA motifs. These are also encountered in the promoter elements of vertebrate snRNA genes, where the first two constitute the distal sequence element (DSE). In all the genes examined so far, the DSE is distant from the PSE and TATA box that compose the basal promoter. However, we observed a fundamental difference in the organization of the H1 RNA and snRNA gene promoters with respect to the relative spacing of the DSE and PSE. Indeed, the H1 promoter is unusually compact, with the octamer motif and Staf binding site adjacent to the PSE and TATA motifs. It thus appears that the human RNase P RNA gene has adopted a unique promoter strategy placing the DSE immediately adjacent to the basal promoter.
Asunto(s)
Endorribonucleasas/biosíntesis , Endorribonucleasas/genética , Regiones Promotoras Genéticas/genética , ARN Polimerasa III/metabolismo , ARN Catalítico/biosíntesis , ARN Catalítico/genética , Transcripción Genética/genética , Proteínas de Xenopus , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Genes Reporteros/genética , Humanos , Ratones , Datos de Secuencia Molecular , Mutación/genética , Oocitos/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa II/metabolismo , ARN Nuclear Pequeño/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Ribonucleasa P , TATA Box/genética , Moldes Genéticos , Transactivadores/química , Transactivadores/metabolismo , Activación Transcripcional , Transfección , Xenopus laevisRESUMEN
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
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
We have recently described the isolation and characterization of bovine cDNA encoding poly(ADP-ribose) glycohydrolase (PARG). We describe here the preparation and characterization of antibodies to PARG. These antibodies have been used to demonstrate the presence of multiple forms of PARG in tissue and cell extracts from bovine, rat, mouse, and insects. Our results indicate that multiple forms of PARG previously reported could result from a single gene. Analysis of PARG in cells in which poly(ADP-ribose) polymerase (PARP) has been genetically inactivated indicates that the cellular content of PARG is regulated independently of PARP.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Animales , Bovinos , Línea Celular , Clonación Molecular , Genotipo , Glicósido Hidrolasas/inmunología , Ratones , Modelos Biológicos , Modelos Genéticos , Ratas , Proteínas Recombinantes de Fusión , Células Tumorales CultivadasRESUMEN
Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.
Asunto(s)
Poli Adenosina Difosfato Ribosa/biosíntesis , Poli(ADP-Ribosa) Polimerasas/fisiología , Células 3T3/efectos de los fármacos , Células 3T3/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Genotipo , Metilnitronitrosoguanidina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutágenos/farmacología , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación/metabolismo , NAD+ Nucleosidasa/antagonistas & inhibidores , NAD+ Nucleosidasa/metabolismo , NAD/análogos & derivados , NAD/farmacología , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Animales , Aplysia/enzimología , Membrana Celular/metabolismo , Perros , Humanos , Isoniazida/farmacología , Células Jurkat , Cinética , Glicoproteínas de Membrana , Ratones , Proteínas Recombinantes/metabolismo , Bazo/enzimología , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Two isomeric azidoadenosyl analogues of adenosine diphosphate (hydroxymethyl)pyrrolidinediol [ADP-HPD; Slama, J. T., et al. (1995) J. Med. Chem. 38, 389-393] were synthesized as photoaffinity labels for poly(ADP-ribose) glycohydrolase. 8-Azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol (8-N3-ADP-HPD) inhibited the enzyme activity by 50% at ca. 1 microM, a concentration 80-fold lower than that where the isomeric 2-azidoadenosine diphosphate (hydroxymethyl)pyrrolidinediol did. [alpha-32P]-8-N3-ADP-HPD was therefore synthesized and used to photoderivatize poly(ADP-ribose) glycohydrolase. Irradiation of recombinant poly(ADP-ribose) glycohydrolase and low concentrations of [alpha-32P]-8-N3-ADP-HPD with short-wave UV light resulted in the covalent incorporation of the photoprobe into the protein, as demonstrated by gel electrophoresis followed by autoradiography or acid precipitation of the protein followed by scintillation counting. No photoincorporation occurred in the absence of UV light. The photoincorporation saturated at low concentrations of the photoprobe and photoprotection was observed in the presence of low concentrations of ADP-HPD, an indication of the specificity of the photoinsertion reaction. These results demonstrate that [alpha-32P]-8-N3-ADP-HPD can be used to specifically covalently photoderivatize the enzyme to characterize the polypetides that constitute the ADP-HPD binding site of poly(ADP-ribose) glycohydrolase. The photoincorporation reaction was further used to determine the ability of ADP-ribose polymers of varying size to compete with [alpha-32P]-8-N3-ADP-HPD for binding to the enzyme. Photoincorporation of [alpha-32P]-8-N3-ADP-HPD was inhibited by 80% in the presence of low concentrations of short, unbranched ADP-ribose oligomers (5-15 ADP-ribose units in length). No similar photoprotection was afforded by the addition of a high-molecular weight highly branched polymer. These results indicate that the photolabel shares a binding site with the short, linear polymer, but not with the long, highly branched polymer.
Asunto(s)
Adenosina Difosfato/análogos & derivados , Glicósido Hidrolasas/metabolismo , Etiquetas de Fotoafinidad/síntesis química , Pirrolidinas/síntesis química , Adenosina Difosfato/síntesis química , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética , Radioisótopos de Fósforo , Etiquetas de Fotoafinidad/metabolismo , Etiquetas de Fotoafinidad/farmacología , Pirrolidinas/metabolismo , Pirrolidinas/farmacologíaRESUMEN
The synthesis and rapid turnover of ADP-ribose polymers is an immediate cellular response to DNA damage. We report here the isolation and characterization of cDNA encoding poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer turnover. PARG was isolated from bovine thymus, yielding a protein of approximately 59 kDa. Based on the sequence of oligopeptides derived from the enzyme, polymerase chain reaction products and partial cDNA clones were isolated and used to construct a putative full-length cDNA. The cDNA of approximately 4.1 kilobase pairs predicted expression of a protein of approximately 111 kDa, nearly twice the size of the isolated protein. A single transcript of approximately 4. 3 kilobase pairs was detected in bovine kidney poly(A)+ RNA, consistent with expression of a protein of 111 kDa. Expression of the cDNA in Escherichia coli resulted in an enzymatically active protein of 111 kDa and an active fragment of 59 kDa. Analysis of restriction endonuclease fragments from bovine DNA by Southern hybridization indicated that PARG is encoded by a single copy gene. Taken together, the results indicate that previous reports of multiple PARGs can be explained by proteolysis of an 111-kDa enzyme. The deduced amino acid sequence of the bovine PARG shares little or no homology with other known proteins. However, it contains a putative bipartite nuclear location signal as would be predicted for a nuclear protein. The availability of cDNA clones for PARG should facilitate structure-function studies of the enzyme and its involvement in cellular responses to genomic damage.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Timo/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Clonación Molecular , ADN/química , ADN/metabolismo , ADN Complementario , Escherichia coli , Genes , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Humanos , Riñón/enzimología , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Cyclic ADP-ribose is a recently discovered metabolite of NAD that appears to function in cellular calcium signaling. The discovery that NAD glycohydrolases are bifunctional enzymes that catalyze both the synthesis and hydrolysis of cyclic ADP-ribose raises many questions concerning the mechanisms by which these enzymes function in calcium signaling. Likewise, the identification of human lymphocyte antigen CD 38 as a bifunctional NAD glycohydrolase raises interesting questions concerning the involvement of cyclic ADP-ribose mediated calcium signaling in immune function. The dementia associated with niacin deficiency has been a long-standing curiosity. This signaling mechanism may resolve questions connecting this vitamin deficiency to central nervous system (CNS) function.