RESUMEN
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
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
The contributions of DNA polymerases alpha, delta, and epsilon to SV40 and nuclear DNA syntheses were evaluated. Proteins were UV-crosslinked to nascent DNA within replicating chromosomes and the photolabelled polymerases were immunopurified. Only DNA polymerases alpha and delta were detectably photolabelled by nascent SV40 DNA, whether synthesized in soluble viral chromatin or within nuclei isolated from SV40-infected cells. In contrast, all three enzymes were photolabelled by the nascent cellular DNA. Mitogenic stimulation enhanced the photolabelling of the polymerases in the alpha>delta>epsilon order of preference. The data agree with the notion that DNA polymerases alpha and delta catalyse the principal DNA polymerisation reactions at the replication fork of SV40 and, perhaps, also of nuclear chromosomes. DNA polymerase epsilon, implicated by others as a cell-cycle checkpoint regulator sensing DNA replication lesions, may be dispensable for replication of the small, fast propagating virus that subverts cell cycle controls.
Asunto(s)
Replicación del ADN , ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Virus 40 de los Simios/genética , Animales , Línea Celular , ADN Polimerasa II , ADN Polimerasa III , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Genoma , Haplorrinos , Mitógenos/farmacología , Virus 40 de los Simios/efectos de la radiación , Rayos UltravioletaRESUMEN
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
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
The 3'-->5' exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3' end of an oligonucleotide with a non-processive mechanism and leaves 5'-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3' end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slowed down when double-stranded region is reached. The preferential removal of a non-complementary 3' end and the nonprocessive mechanism are consistent with anticipated proofreading function. In addition to the 3'-->5' exonuclease activity, an 5'-->3' exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential component for the action of this enzyme in vivo. Contrary to the 3'-->5' exonuclease activity, the 5'-->3' exonuclease is separable from the polymerase activity.