RESUMEN
The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase epsilon (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase alpha, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , ADN Polimerasa II/genética , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genética , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , División Celular , Medio de Cultivo Libre de Suero , ADN/genética , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Inducción Enzimática , Exones/genética , Células HeLa , Humanos , Intrones/genética , Sustancias Macromoleculares , Modelos Genéticos , Datos de Secuencia Molecular , Mutación/genética , Factores de Transcripción NFI , Ensayos de Protección de Nucleasas , Unión Proteica , ARN Mensajero/análisis , ARN Mensajero/genética , Proteína 1 de Unión a Retinoblastoma , Transcripción Genética/genéticaRESUMEN
In mammalian cells, DNA double-strand breaks are repaired mainly by non-homologous end joining, which modifies and ligates two DNA ends without requiring extensive base pairing interactions for alignment. We investigated the role of DNA polymerases in DNA-PK-dependent end joining of restriction-digested plasmids in vitro and in vivo. Rejoining of DNA blunt ends as well as those with partially complementary 5' or 3' overhangs was stimulated by 20-53% in HeLa cell-free extracts when dNTPs were included, indicating that part of the end joining is dependent on DNA synthesis. This DNA synthesis-dependent end joining was sensitive to aphidicolin, an inhibitor of alpha-like DNA polymerases. Furthermore, antibodies that neutralize the activity of DNA polymerase alpha were found to strongly inhibit end joining in vitro, whereas neutralizing antibodies directed against DNA polymerases beta and epsilon did not. DNA sequence analysis of end joining products revealed two prominent modes of repair, one of which appeared to be dependent on DNA synthesis. Identical products of end joining were recovered from HeLa cells after transfection with one of the model substrates, suggesting that the same end joining mechanisms also operate in vivo. Fractionation of cell extracts to separate PCNA as well as depletion of cell extracts for PCNA resulted in a moderate but significant reduction in end joining activity, suggesting a potential role in a minor repair pathway.
Asunto(s)
ADN Polimerasa I/metabolismo , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN , ADN/metabolismo , Recombinación Genética/genética , Anticuerpos/inmunología , Anticuerpos/farmacología , Afidicolina/farmacología , Secuencia de Bases , Southern Blotting , Extractos Celulares , ADN/biosíntesis , ADN/genética , Daño del ADN/genética , ADN Polimerasa I/antagonistas & inhibidores , ADN Polimerasa I/inmunología , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN , Células HeLa , Humanos , Proteínas Nucleares , Nucleótidos/metabolismo , Plásmidos/biosíntesis , Plásmidos/genética , Plásmidos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética/efectos de los fármacos , Homología de Secuencia , Especificidad por Sustrato , TransfecciónRESUMEN
Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Daño del ADN , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN , Proteínas de Drosophila , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Transglutaminasas , Animales , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Mapeo Cromosómico , Clonación Molecular , Citoplasma/metabolismo , ADN Polimerasa II/metabolismo , ADN Complementario/metabolismo , Drosophila , Ecdisona/metabolismo , Proteínas Fúngicas/química , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Ratones , Proteínas Nucleares , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Fase S , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Transducción de Señal , Factores de Tiempo , Células Tumorales Cultivadas , Técnicas del Sistema de Dos HíbridosRESUMEN
The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.
Asunto(s)
Trifosfato de Arabinofuranosil Citosina/farmacología , ADN Polimerasa III/antagonistas & inhibidores , ADN Polimerasa I/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Línea Celular , ADN/biosíntesis , ADN/efectos de los fármacos , ADN Polimerasa I/metabolismo , ADN Polimerasa III/metabolismo , Células HeLa , Humanos , Virus 40 de los Simios/fisiologíaRESUMEN
In eukaryotic cells, DNA polymerases are required to maintain the integrity of the genome during processes, such as DNA replication, various DNA repair events, translesion DNA synthesis, DNA recombination, and also in regulatory events, such as cell cycle control and DNA damage checkpoint function. In the last two years, the number of known DNA polymerases has increased to at least nine (called alpha, beta, gamma, delta, epsilon, zeta, eta, t and iota), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Células Eucariotas/enzimología , Nucleotidiltransferasas , Proteínas de Saccharomyces cerevisiae , Animales , ADN/biosíntesis , ADN Polimerasa III/metabolismo , ADN Polimerasa beta/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , ADN Polimerasa iota , ADN Polimerasa thetaRESUMEN
During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.
Asunto(s)
Quinasas Ciclina-Dependientes , Reparación del ADN/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción TFII , Factores de Transcripción/metabolismo , Células HeLa , Humanos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Transcripción TFIIH , Factores de Transcripción/química , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.
Asunto(s)
ADN Polimerasa II/metabolismo , Replicación del ADN , ADN Viral/biosíntesis , Virus 40 de los Simios/genética , Animales , Anticuerpos/inmunología , Bromodesoxiuridina/metabolismo , Dominio Catalítico , Bovinos , Línea Celular , ADN Polimerasa II/antagonistas & inhibidores , ADN Polimerasa II/inmunología , Fibroblastos/citología , Células HeLa , Humanos , Pruebas de Neutralización , Conejos , Virus 40 de los Simios/fisiología , Replicación ViralRESUMEN
The cDNA and the gene for the mouse DNA polymerase epsilon catalytic subunit were cloned. The deduced protein sequence shows remarkable evolutionary conservation in DNA polymerase epsilon family. However, several conserved elements involved in template-primer binding differ from those of other class B polymerases. This is likely to reflect a distinctive function of the enzyme. The gene that was assigned to chromosome 5 region E3-E5, consists of 49 exons and has a non-conforming splice site in the junction of exon and intron 13. A CpG island covers the promoter region which contains several putative consensus elements critical for S phase upregulated and serum responsive promoters.
Asunto(s)
ADN Polimerasa II/química , ADN Polimerasa II/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , ADN Complementario/química , Exones , Intrones , Ratones , Datos de Secuencia Molecular , Proteínas de Unión a Poli-ADP-RibosaRESUMEN
DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.
Asunto(s)
ADN Polimerasa II/metabolismo , Reparación del ADN , Replicación del ADN , Levaduras/genética , Alelos , Dominio Catalítico , ADN Polimerasa II/química , ADN de Hongos/análisis , Regulación Fúngica de la Expresión Génica , Mutagénesis/fisiología , Regiones Promotoras Genéticas/fisiología , Estructura Terciaria de Proteína , Levaduras/citología , Levaduras/enzimologíaRESUMEN
The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters.
Asunto(s)
Empalme Alternativo , Dominio Catalítico/genética , ADN Polimerasa II/genética , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cromosomas Humanos Par 12/genética , Clonación Molecular , Codón Iniciador/genética , Islas de CpG/genética , Exones/genética , Dosificación de Gen , Biblioteca de Genes , Células HeLa , Humanos , Intrones/genética , Datos de Secuencia Molecular , Poli A/genética , Proteínas de Unión a Poli-ADP-Ribosa , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , TATA Box/genéticaAsunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , ADN Polimerasa Dirigida por ADN/clasificación , Humanos , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Pyrococcus furiosus/enzimología , Pyrococcus furiosus/genética , Homología de Secuencia de AminoácidoRESUMEN
Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function. cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned. The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical. The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae. The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication. The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha. The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.
Asunto(s)
ADN Polimerasa II/química , ADN Polimerasa II/genética , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cromosomas Humanos Par 14/genética , Clonación Molecular , ADN Complementario/genética , Humanos , Ratones , Datos de Secuencia Molecular , Peso Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.
Asunto(s)
Mama/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Complejos Multienzimáticos/metabolismo , Células Cultivadas , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa I/metabolismo , ADN Primasa/metabolismo , Replicación del ADN , Femenino , Humanos , Mapeo Peptídico , Poli(ADP-Ribosa) Polimerasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/farmacología , Unión ProteicaRESUMEN
Previous studies on DNA polymerase epsilon indicate that this enzyme is involved in replication of chromosomal DNA. In this study, we examined the expression of DNA polymerases alpha, delta, and epsilon during mouse testis development and germ cell differentiation. The steady-state levels of mRNAs encoding DNA polymerase epsilon and the recombination enzyme Rad51 remained constant during testis development, whereas the mRNA levels of DNA polymerases alpha and delta declined from birth until sexual maturity. Immunohistochemical staining methods, using a stage-specific model of the seminiferous epithelium, revealed dramatic differences between DNA polymerase alpha and epsilon distribution. As expected, DNA polymerase alpha and proliferating cell nuclear antigen showed relatively strong immunostaining in mitotically proliferating spermatogonia and even stronger staining in preleptotene cells undergoing meiotic DNA replication. The distribution of Rad51 was similar, but there was a dramatic peak in late pachytene cells. In contrast, DNA polymerase epsilon was detectable in mitotically proliferating spermatogonia but not in the early stages of meiotic prophase. However, DNA polymerase epsilon reappeared in late pachytene cells and remained through the two meiotic divisions, and was present in haploid spermatids up to the stage at which the flagellum starts developing. Overall, the results suggest that DNA polymerase epsilon functions in mitotic replication, in the completion of recombination in late pachytene cells, and in repair of DNA damage in round spermatids. In contrast, DNA polymerases alpha and delta appear to be involved in meiotic DNA synthesis, which occurs early in meiotic prophase, in addition to functioning in DNA replication in proliferating spermatogonia.
Asunto(s)
ADN Polimerasa II/metabolismo , Replicación del ADN , Espermatogénesis/fisiología , Espermatozoides/enzimología , Testículo/crecimiento & desarrollo , Animales , ADN Polimerasa I/análisis , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa II/análisis , ADN Polimerasa II/genética , ADN Polimerasa III/análisis , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/análisis , Expresión Génica , Masculino , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Antígeno Nuclear de Célula en Proliferación/análisis , ARN Mensajero/análisis , Recombinasa Rad51 , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Epitelio Seminífero/citología , Espermatozoides/fisiología , Testículo/citologíaRESUMEN
In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Replicación del ADN/fisiología , ADN de Neoplasias/biosíntesis , Animales , Antígenos Transformadores de Poliomavirus/aislamiento & purificación , Neoplasias de la Mama/química , Carcinoma Ductal de Mama/química , ADN Primasa , ADN Polimerasa Dirigida por ADN/análisis , Femenino , Humanos , Inmunohistoquímica , Sustancias Macromoleculares , Ratones , Ratones Desnudos , Complejos Multiproteicos , Trasplante de Neoplasias , Antígeno Nuclear de Célula en Proliferación/análisis , ARN Nucleotidiltransferasas/análisis , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Double-stranded RNA bacteriophage phi 6 has an envelope surrounding the nucleocapsid (NC). The NC is composed of a surface protein, P8, and proteins P1, P2, P4, and P7, which form a dodecahedral polymerase complex enclosing the segmented viral genome. Empty polymerase complex particles (procapsids) package positive-sense viral single-stranded RNAs provided that energy is available in the form of nucleoside triphosphates (NTPs). Photoaffinity labelling of both the NC and the procapsid has earlier been used to show that ATP binds to protein P4 and that the NC hydrolyzes NTPs. Using the NC and the NC core particles (NCs lacking surface protein P8) and purified protein P4, we demonstrate here that multimeric P4 is the active NTPase. Isolation of multimeric P4 is successful only in the presence of NTPs. The activity of P4 is the same in association with the viral particles as it is in pure form. P4 is an unspecific NTPase hydrolyzing ribo-NTPs, deoxy NTPs, and dideoxy NTPs to the corresponding nucleoside diphosphates. The Km of the reaction for ATP, GTP, and UTP is around 0.2 to 0.3 mM. The NTP hydrolysis by P4 absolutely requires residual amounts of Mg2+ ions and is greatly activated when the Ca2+ concentration reaches 0.5 mM. Competition experiments indicate that Mg2+ and Ca2+ ions have approximately equal binding affinities for P4. They might compete for a common binding site. The nucleotide specificity and enzymatic properties of the P4 NTPase are similar to the NTP hydrolysis reaction conditions needed to translocate and condense the viral positive-sense RNAs to the procapsid particle.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Bacteriófago phi 6/enzimología , Calcio/farmacología , Ácido Anhídrido Hidrolasas/aislamiento & purificación , Bacteriófago phi 6/genética , Cápside/química , Cápside/aislamiento & purificación , Cápside/metabolismo , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Genoma Viral , Cinética , Magnesio/farmacología , Nucleósido-Trifosfatasa , Especificidad por Sustrato , TermodinámicaRESUMEN
In order to shed light on the role of mammalian DNA polymerase epsilon we studied the expression of mRNA for the human enzyme during cell proliferation and during the cell cycle. Steady-state levels of mRNA encoding DNA polymerase epsilon were elevated dramatically when quiescent (G0) cells were stimulated to proliferate (G1/S) in a similar manner to those of DNA polymerase alpha. Message levels of DNA polymerase beta were unchanged in similar experiments. The concentration of immunoreactive DNA polymerase epsilon was also much higher in extracts from proliferating tissues than in those from non-proliferating or slowly proliferating tissues. The level of DNA polymerase epsilon mRNA in actively cycling cells synchronized with nocodazole and in cells fractionated by counterflow centrifugal elutriation showed weaker variation, being at its highest at the G1/S stage boundary. The results presented strongly suggest that mammalian DNA polymerase epsilon is involved in the replication of chromosomal DNA and/or in a repair process that may be substantially activated during the replication of chromosomal DNA. A hypothetical role for DNA polymerase epsilon in a repair process coupled to replication is discussed.
Asunto(s)
División Celular , ADN Polimerasa Dirigida por ADN/genética , Expresión Génica , Ciclo Celular , ADN Polimerasa I/genética , ADN Polimerasa II/genética , Reparación del ADN/fisiología , Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Fase G1 , Células HeLa , Humanos , Nocodazol/farmacología , ARN Mensajero/metabolismo , Fase de Descanso del Ciclo Celular , Fase SRESUMEN
Monoclonal antibodies raised against the N-terminal half of human DNA polymerase epsilon bind both to a large > 200 kDa form of DNA polymerase epsilon from HeLa cells and to a small 140 kDa form (DNA polymerase epsilon*) from calf thymus, while antibody against the C-terminal half binds to DNA polymerase epsilon but does not bind to DNA polymerase epsilon*. These results indicate that the two enzymes have common structural motifs in their N-terminal halves, and that DNA polymerase epsilon* is very likely derived from DNA polymerase epsilon by removal of its C-terminal half. DNA polymerase epsilon as well as DNA polymerase epsilon* was detected in extracts from cells of numerous eukaryotic species from yeast to human. The results indicate that DNA polymerase epsilon and its tendency to occur in a smaller form, DNA polymerase epsilon*, are evolutionarily highly conserved and that DNA polymerase epsilon may occur universally in proliferating eukaryotic cells.
Asunto(s)
ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/química , Animales , Anticuerpos Monoclonales/inmunología , Evolución Biológica , Western Blotting , Bovinos , División Celular , ADN Polimerasa II , ADN Polimerasa Dirigida por ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Eucariotas/enzimología , Células HeLa/enzimología , Humanos , Fragmentos de Péptidos/inmunología , Timo/enzimologíaRESUMEN
DNA polymerase epsilon [DNA pol epsilon (EC 2.7.7.7)] is one of the four nuclear DNA polymerases in eukaryotic cells. The mammalian enzyme is involved in DNA repair and possibly also in replication of chromosomal DNA. The gene encoding pol epsilon (POLE) was assigned to human and rat chromosomes 12 by Southern blot analysis of genomic DNA from mouse-human and mouse-rat somatic cell hybrid panels using human cDNA probe. The human gene was then regionally localized to band 12q24.3 by fluorescence in situ hybridization of metaphase spreads of chromosomes from human lymphocytes using genomic DNA probe. POLE is closely linked to HNF1A, a gene encoding a liver-enriched transcription factor, HNF1 alpha. The two genes thus define a new synteny group retained on human and rat chromosomes 12. Another gene mapping to the same or close-by region as human POLE is a gene for inherited disorder tuberous sclerosis 3, TSC3.
Asunto(s)
Mapeo Cromosómico , Cromosomas Humanos Par 12 , ADN Polimerasa Dirigida por ADN/genética , Animales , ADN Polimerasa II , Reparación del ADN , Replicación del ADN , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , RatasRESUMEN
The cDNA encoding the catalytic polypeptide of human DNA polymerase epsilon was cloned. The deduced amino acid sequence reveals that the catalytic polypeptide is 2257 amino acids in length and its calculated molecular mass is 258 kDa. A single RNA message of 7.5 kilobases was recognized by isolated cDNA clones. The identity of the cDNA was verified by direct amino acid sequencing of tryptic fragments derived from the catalytic polypeptide of the HeLa DNA polymerase epsilon. The primary structure comparison with multiple DNA polymerases indicates that human DNA polymerase epsilon catalytic polypeptide is a homolog of the yeast Saccharomyces cerevisiae DNA polymerase II catalytic polypeptide. The proteins are 39% identical. In the region containing known DNA polymerase consensus motifs, the identity is 63%. The expression of the mRNA encoding DNA polymerase epsilon is strongly dependent on cell proliferation.