RESUMEN

Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.

Asunto(s)

Apoptosis/fisiología , Roturas del ADN de Cadena Simple , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Apraxias/metabolismo , Apraxias/patología , Apraxias/fisiopatología , Ataxia/metabolismo , Ataxia/patología , Ataxia/fisiopatología , Western Blotting , Camptotecina/farmacología , Células Cultivadas , Daño del ADN , Reparación del ADN , Etopósido/farmacología , Femenino , Citometría de Flujo , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Potencial de la Membrana Mitocondrial/efectos de la radiación , Metilnitronitrosoguanidina/farmacología , Mitomicina/farmacología , Poli(ADP-Ribosa) Polimerasa-1 , Radiación Ionizante , Especies de Nitrógeno Reactivo/metabolismo

RESUMEN

By screening for Arabidopsis genes activated by ionising radiation (IR)-induced DNA damage, we have isolated a cDNA hybridising with a 3.2-kb mRNA that accumulates rapidly and strongly in irradiated cell suspensions or whole plants. The cDNA codes for a 110-kDa protein that is highly homologous to the 116-kDa vertebrate poly(ADP-ribose) polymerase (PARP-1). It is recognised by a human anti-PARP-1 antibody, binds efficiently to DNA strand interruptions in vitro, and catalyses DNA damage-dependent (ADP-ribose) polymer synthesis. We have named this protein AtPARP-1. We have also extended our observations to the Arabidopsis app (AtPARP-2) gene, demonstrating for the first time that IR-induced DNA strand interruptions induce rapid and massive accumulation of AtPARP-1 and AtPARP-2 transcripts, whereas dehydration and cadmium preferentially induce the accumulation of AtPARP-2 transcripts. The IR-induced PARP gene expression seen in Arabidopsis is in striking contrast to the post-translational activation of the PARP-1 protein that is associated with genotoxic stress in animal cells. AtPARP-1 transcripts accumulate in all plant organs after exposure to ionising radiation, but this is followed by an increase in AtPARP-1 protein levels only in tissues that contain large amounts of actively dividing cells. This cell-type specific accumulation of AtPARP-1 protein in response to DNA damage is compatible with a role for the AtPARP-1 protein in the maintenance of DNA integrity during replication, similar to the role of "guardian of the genome" attributed to its animal counterpart.

Asunto(s)

Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Poli(ADP-Ribosa) Polimerasas/genética , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Arabidopsis/efectos de la radiación , Dominio Catalítico , Radioisótopos de Cobalto , Secuencia Conservada , Rayos gamma , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Humanos , Isoenzimas/química , Isoenzimas/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Poli(ADP-Ribosa) Polimerasas/química , ARN Mensajero/genética , ARN de Planta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Vertebrados/genética , Dedos de Zinc

RESUMEN

Poly(ADP-ribose) polymerase (PARP)-1, a detector of single-strand breaks, plays a key role in the cellular response to DNA damage. PARP-1-deficient mice are hypersensitive to genotoxic agents and display genomic instability due to a DNA repair defect in the base excision repair pathway. A previous report suggested that PARP-1-deficient mice also had a severe telomeric dysfunction consisting of telomere shortening and increased end-to-end fusions (d'Adda di Fagagna, F., M.P. Hande, W.-M. Tong, P.M. Lansdorp, Z.-Q. Wang, and S.P. Jackson. 1999. NAT: Genet. 23:76-80). In contrast to that, and using a panoply of techniques, including quantitative telomeric (Q)-FISH, we did not find significant differences in telomere length between wild-type and PARP-1(-/)- littermate mice or PARP-1(-/)- primary cells. Similarly, there were no differences in the length of the G-strand overhang. Q-FISH and spectral karyotyping analyses of primary PARP-1(-/)- cells showed a frequency of 2 end-to-end fusions per 100 metaphases, much lower than that described previously (d'Adda di Fagagna et al., 1999). This low frequency of end-to-end fusions in PARP-1(-/)- primary cells is accordant with the absence of severe proliferative defects in PARP-1(-/)- mice. The results presented here indicate that PARP-1 does not play a major role in regulating telomere length or in telomeric end capping, and the chromosomal instability of PARP-1(-/)- primary cells can be explained by the repair defect associated to PARP-1 deficiency. Finally, no interaction between PARP-1 and the telomerase reverse transcriptase subunit, Tert, was found using the two-hybrid assay.

Asunto(s)

Cromosomas/química , Cromosomas/ultraestructura , Poli(ADP-Ribosa) Polimerasas/genética , ARN , Telómero/química , Telómero/ultraestructura , Animales , Células de la Médula Ósea/metabolismo , División Celular , Células Cultivadas , ADN Complementario/metabolismo , Proteínas de Unión al ADN , Genotipo , Heterocigoto , Hibridación Fluorescente in Situ , Cariotipificación , Ratones , Ratones Transgénicos , Poli(ADP-Ribosa) Polimerasas/fisiología , Bazo/citología , Telomerasa/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

PARP-1-deficient mice display a severe defect in the base excision repair pathway leading to radiosensitivity and genomic instability. They are protected against necrosis induced by massive oxidative stress in various inflammatory processes. Mice lacking p53 are highly predisposed to malignancy resulting from defective cell cycle checkpoints, resistance to DNA damage-induced apoptosis as well as from upregulation of the iNOS gene resulting in chronic oxidative stress. Here, we report the generation of doubly null mutant mice. We found that tumour-free survival of parp-1(-/-)p53(-/-) mice increased by 50% compared with that of parp- 1(+/+)p53(-/-) mice. Tumour formation in nude mice injected with oncogenic parp-1(-/-)p53(-/-) fibroblasts was significantly delayed compared with parp-1(+/+)p53(-/-) cells. Upon gamma-irradiation, a partial restoration of S-phase radiosensitivity was found in parp-1(-/-)p53(-/-) primary fibroblasts compared with parp-1(+/+)p53(-/-) cells. In addition, iNOS expression and nitrite release were dramatically reduced in the parp-1(-/-)p53(-/-) mice compared with parp-1(+/+)p53(-/-) mice. The abrogation of the oxydated status of p53(-/-) cells, due to the absence of parp-1, may be the cause of the delay in the onset of tumorigenesis in parp-1(-/-)p53(-/-) mice.

Asunto(s)

Reparación del ADN , Genes p53 , Genes ras , Neoplasias Experimentales/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Ciclo Celular/fisiología , Ciclo Celular/efectos de la radiación , Transformación Celular Neoplásica , Células Cultivadas , Cruzamientos Genéticos , Supervivencia sin Enfermedad , Femenino , Fibroblastos/fisiología , Fibroblastos/efectos de la radiación , Fibroblastos/trasplante , Rayos gamma , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Ratones Desnudos , Micronúcleos con Defecto Cromosómico/genética , Micronúcleos con Defecto Cromosómico/patología , Micronúcleos con Defecto Cromosómico/ultraestructura , Neoplasias Experimentales/patología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética

RESUMEN

PARP-1 and ATM are both involved in the response to DNA strand breaks, resulting in induction of a signaling network responsible for DNA surveillance, cellular recovery, and cell survival. ATM interacts with double-strand break repair pathways and induces signals resulting in the control of the cell cycle-coupled checkpoints. PARP-1 acts as a DNA break sensor in the base excision repair pathway of DNA. Mice with mutations inactivating either protein show radiosensitivity and high radiation-induced chromosomal aberration frequencies. Embryos carrying double mutations of both PARP-1 and Atm genes were generated. These mutant embryos show apoptosis in the embryo but not in extraembryonic tissues and die at embryonic day 8.0, although extraembryonic tissues appear normal for up to 10.5 days of gestation. These results reveal a functional synergy between PARP-1 and ATM during a period of embryogenesis when cell cycle checkpoints are not active and the embryo is particularly sensitive to DNA damage. These results suggest that ATM and PARP-1 have synergistic phenotypes due to the effects of these proteins on signaling DNA damage and/or on distinct pathways of DNA repair.

Asunto(s)

Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas/genética , Proteínas/fisiología , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Proteínas de Ciclo Celular , Muerte Celular , División Celular , Supervivencia Celular , Aberraciones Cromosómicas , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Embrión de Mamíferos/metabolismo , Genotipo , Heterocigoto , Ratones , Microscopía Electrónica , Modelos Biológicos , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas , Reacción en Cadena de la Polimerasa , Tolerancia a Radiación , Transducción de Señal , Factores de Tiempo , Proteínas Supresoras de Tumor

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 P

RESUMEN

Poly (ADP-ribose) polymerase (113 kDa; PARP-1) is a constitutive factor of the DNA damage surveillance network developed by the eukaryotic cell to cope with the numerous environmental and endogenous genotoxic agents. This enzyme recognizes and is activated by DNA strand breaks. This original property plays an essential role in the protection and processing of the DNA ends as they arise in DNA damage that triggers the base excision repair (BER) pathway. The generation, by homologous recombination, of three independent deficient mouse models have confirmed the caretaker function of PARP-1 in mammalian cells under genotoxic stress. Unexpectedly, the knockout strategy has revealed the instrumental role of PARP-1 in cell death after ischemia-reperfusion injury and in various inflammation process. Moreover, the residual PARP activity found in PARP-1 deficient cells has been recently attributed to a novel DNA damage-dependent poly ADP-ribose polymerase (62 kDa; PARP-2), another member of the expanding PARP family that, on the whole, appears to be involved in the genome protection. The present review summarizes the recent data obtained with the three PARP knockout mice in comparison with the chemical inhibitor approach.

Asunto(s)

Eliminación de Gen , Ratones Noqueados , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Animales , Daño del ADN/genética , Reparación del ADN/genética , Diabetes Mellitus Tipo 1/genética , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Fenotipo , Poli(ADP-Ribosa) Polimerasas/deficiencia , Poli(ADP-Ribosa) Polimerasas/genética

RESUMEN

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.

Asunto(s)

Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Células Cultivadas , ADN Polimerasa beta/metabolismo , Cartilla de ADN , Ratones , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética

RESUMEN

PURPOSE: To determine whether DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP-1) are involved in eliciting the rapid fluctuations of radiosensitivity that have been observed when cells are exposed to short pulses of ionizing radiation. MATERIALS AND METHODS: The effect of DNA-PK and PARP-1 inhibitors on the survival of cells to split-dose irradiation was investigated using Chinese hamster V79 fibroblasts and human carcinoma SQ-20B cells. The responses of PARP-1 proficient and PARP-1 knockout mouse 3T3 fibroblasts were compared in a similar split-dose assay. RESULTS: Inactivation of DNA-PK by wortmannin potentiated radiation-induced cell kill but it did not alter the oscillatory, W-shaped pattern of early radiation response. In contrast, oscillatory radiation response was abolished by 3-aminobenzamide, a reversible inhibitor of enzymes containing a PARP catalytic domain. The oscillatory response was also lacking in PARP-1 knockout mouse 3T3 fibroblasts. CONCLUSION: The results show that PARP-1 plays a key role in the earliest steps of cell response to ionizing radiation with clonogenic ability or growth as endpoint. It is hypothesized that rapid poly(ADP-ribosylation) of target proteins, or recruitment of repair proteins by activated PARP-1 at the sites of DNA damage, bring about rapid chromatin remodelling that may affect the incidence of chromosomal damage upon re-irradiation.

Asunto(s)

Proteínas de Unión al ADN , Poli(ADP-Ribosa) Polimerasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Células 3T3 , Androstadienos/farmacología , Animales , Benzamidas/farmacología , Dominio Catalítico/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Cromatina/efectos de la radiación , Cricetinae , Proteína Quinasa Activada por ADN , Relación Dosis-Respuesta en la Radiación , Inhibidores Enzimáticos/farmacología , Fibroblastos/efectos de la radiación , Rayos gamma/efectos adversos , Humanos , Ratones , Proteínas Nucleares , Radiación Ionizante , Factores de Tiempo , Células Tumorales Cultivadas , Wortmanina

RESUMEN

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-kappaB in macrophages from PARP-1(+/+) but not from PARP-1(-/-) mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process.

Asunto(s)

Proteínas I-kappa B , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasas/fisiología , Choque Séptico/inmunología , Células 3T3 , Animales , Aorta , Transporte Biológico , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Interleucina-6/biosíntesis , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Poli(ADP-Ribosa) Polimerasas/genética , Choque Séptico/fisiopatología , Factor de Transcripción ReIA , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

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 Xenopus

RESUMEN

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 Secuencia

RESUMEN

A reliable ELISA for screening large numbers of poly(ADP-ribose) polymerase (PARP) inhibitors is described. The test is based upon the drop in PARP activity estimated by the decrease in poly(ADP-ribose) synthesis in the presence of inhibitor. This ELISA is easy to perform, rapid, and specific. It is extremely sensitive because a clear inhibition of the total reaction could be visualized with molecules used in the nanomolar range. The assay uses no radioactivity, and automation is possible with robots for large-scale investigations. This test is of great interest for the screening of chemical libraries and the discovery of new inhibitors (and possibly activators) of PARP. Such molecules have important applications in all abnormal situations involving DNA damage and oxidative stress, such as cancer, autoimmunity, diabetes, myocardial dysfunctions, certain infections, aging, and radiation/chemical exposure.

Asunto(s)

Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Apoptosis , Automatización , Daño del ADN , Reparación del ADN , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Recombinantes/antagonistas & inhibidores , Sensibilidad y Especificidad

RESUMEN

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 Hermanas

Asunto(s)

Apoptosis/fisiología , Daño del ADN/fisiología , Reparación del ADN/fisiología , Poli(ADP-Ribosa) Polimerasas/fisiología , Animales , Apoptosis/genética , Caspasas/metabolismo , Reparación del ADN/genética , Activación Enzimática , Humanos , Ratones , Ratones Noqueados , Necrosis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

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/genética

RESUMEN

Poly(ADP-ribose) polymerase (PARP) is a constitutive factor of the DNA damage surveillance network in dividing cells. Based on its capacity to bind to DNA strand breaks, PARP plays a regulatory role in their resolution in vivo. ATM belongs to a large family of proteins involved in cell cycle progression and checkpoints in response to DNA damage. Both proteins may act as sensors of DNA damage to induce multiple signalling pathways leading to activation of cell cycle checkpoints and DNA repair. To determine a possible relationship between PARP and ATM, we examined the PARP response in an ATM-null background. We demonstrated that ATM deficiency does not affect PARP activity in human cell lines or Atm-deficient mouse tissues, nor does it alter PARP activity induced by oxidative damage or gamma-irradiation. Our results support a model in which PARP and ATM could be involved in distinct pathways, both effectors transducing the damage signal to cell cycle regulators.

Asunto(s)

Ataxia Telangiectasia/enzimología , Fibroblastos/enzimología , Linfocitos/enzimología , Poli(ADP-Ribosa) Polimerasas/análisis , Proteínas Serina-Treonina Quinasas , Proteínas/fisiología , Animales , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Línea Celular Transformada , Células Cultivadas , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Rayos gamma , Humanos , Peróxido de Hidrógeno/farmacología , Linfocitos/efectos de los fármacos , Linfocitos/efectos de la radiación , Masculino , Ratones , Ratones Noqueados , Estrés Oxidativo , Proteínas/genética , Transducción de Señal , Bazo/enzimología , Testículo/enzimología , Proteínas Supresoras de Tumor

RESUMEN

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.

Asunto(s)

Apoptosis/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Alquilantes/farmacología , Animales , Línea Celular Transformada , Núcleo Celular/enzimología , Núcleo Celular/metabolismo , Citoplasma/enzimología , Citoplasma/metabolismo , Hidrólisis , Ratones , Mutagénesis Sitio-Dirigida , Poli(ADP-Ribosa) Polimerasas/genética , Receptor fas/metabolismo

RESUMEN

Recent studies with poly(ADP-ribose) polymerase (PARP)-deficient mice have highlighted the role of this enzyme in genomic stability and response to various genomic insults. In the absence of DNA damaging treatment, we report here that a PARP-deficient cell line (PARP-/-) established from knockout mice displays a decrease in topoisomerase II (topo II) activity as measured by decatenation of kinetoplast DNA. Immunoblotting of whole and nuclear cell extracts showed that reduced activity was associated with decreased amount of the 180 kDa topo IIbeta protein but not of the 170 kDa topo IIalpha. The decreased topo IIbeta expression did not stem from transcriptional regulation of gene expression since levels of topo IIbeta mRNA were similar in PARP (-/-) compared with the parental PARP (+/+) cells. The decreased topo II activity was associated with cell resistance to VP16, a topo II inhibitor. These observations indicate that PARP may play a role in the stabilization and/or distribution of topo IIbeta.

Asunto(s)

ADN-Topoisomerasas de Tipo II/deficiencia , Poli(ADP-Ribosa) Polimerasas/deficiencia , Animales , Línea Celular , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN , Resistencia a Medicamentos , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Noqueados , Peso Molecular , Poli(ADP-Ribosa) Polimerasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Inhibidores de Topoisomerasa II

RESUMEN

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Asunto(s)

Linfocitos B/inmunología , Reparación del ADN/fisiología , Reordenamiento Génico de Linfocito B , Genes de Inmunoglobulinas , Memoria Inmunológica/inmunología , Mutación , Animales , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa