RESUMEN
Varicella zoster virus (VZV) is a pathogenic human herpes virus which causes varicella as a primary infection, following which it becomes latent in peripheral autonomic, sensory, and cranial nerve ganglionic neurons from where it may reactivate after decades to cause herpes zoster. VZV reactivation may also cause a wide spectrum of neurological syndromes, in particular, acute encephalitis and vasculopathy. While there is potentially a large number of coding viral mutations that might predispose certain individuals to VZV infections, in practice, a variety of host factors are the main determinants of VZV infection, both disseminated and specifically affecting the nervous system. Host factors include increasing age with diminished cell-mediated immunity to VZV, several primary immunodeficiency syndromes, secondary immunodeficiency syndromes, and drug-induced immunosuppression. In some cases, the molecular immunological basis underlying the increased risk of VZV infections has been defined, in particular, the role of POL III mutations, but in other cases, the mechanisms have yet to be determined. The role of immunization in immunosuppressed individuals as well as its possible efficacy in preventing both generalized and CNS-specific infections will require further investigation to clarify in such patients.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Encefalitis por Varicela Zóster/virología , Herpesvirus Humano 3/patogenicidad , Interacciones Huésped-Patógeno/inmunología , Huésped Inmunocomprometido , Sistema Nervioso/virología , Enfermedades de Inmunodeficiencia Primaria/virología , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/inmunología , ADN Polimerasa III/genética , ADN Polimerasa III/inmunología , Encefalitis por Varicela Zóster/complicaciones , Encefalitis por Varicela Zóster/genética , Encefalitis por Varicela Zóster/inmunología , Expresión Génica , Herpesvirus Humano 3/inmunología , Interacciones Huésped-Patógeno/genética , Humanos , Inmunidad Celular , Inmunosupresores/efectos adversos , Linfocitos/inmunología , Linfocitos/patología , Linfocitos/virología , Mutación , Sistema Nervioso/inmunología , Sistema Nervioso/patología , Enfermedades de Inmunodeficiencia Primaria/complicaciones , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/inmunología , Latencia del Virus/inmunologíaRESUMEN
BACKGROUND: Mutations affecting DNA polymerases have been implicated in genomic instability and cancer development, but the mechanisms by which they can affect the immune system remain largely unexplored. OBJECTIVE: We sought to establish the role of DNA polymerase δ1 catalytic subunit (POLD1) as the cause of a primary immunodeficiency in an extended kindred. METHODS: We performed whole-exome and targeted gene sequencing, lymphocyte characterization, molecular and functional analyses of the DNA polymerase δ (Polδ) complex, and T- and B-cell antigen receptor repertoire analysis. RESULTS: We identified a missense mutation (c. 3178C>T; p.R1060C) in POLD1 in 3 related subjects who presented with recurrent, especially herpetic, infections and T-cell lymphopenia with impaired T-cell but not B-cell proliferation. The mutation destabilizes the Polδ complex, leading to ineffective recruitment of replication factor C to initiate DNA replication. Molecular dynamics simulation revealed that the R1060C mutation disrupts the intramolecular interaction between the POLD1 CysB motif and the catalytic domain and also between POLD1 and the Polδ subunit POLD2. The patients exhibited decreased numbers of naive CD4 and especially CD8 T cells in favor of effector memory subpopulations. This skewing was associated with oligoclonality and restricted T-cell receptor ß-chain V-J pairing in CD8+ but not CD4+ T cells, suggesting that POLD1R1060C differentially affects peripheral CD8+ T-cell expansion and possibly thymic selection. CONCLUSION: These results identify gene defects in POLD1 as a novel cause of T-cell immunodeficiency.
Asunto(s)
ADN Polimerasa III , Mutación con Pérdida de Función , Mutación Missense , Inmunodeficiencia Combinada Grave , Adolescente , Secuencias de Aminoácidos , Preescolar , ADN Polimerasa III/genética , ADN Polimerasa III/inmunología , Femenino , Células HEK293 , Humanos , Dominios Proteicos , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Secuenciación del ExomaRESUMEN
Replication through a diverse array of DNA lesions occurs by the sequential action of two translesion synthesis (TLS) DNA polymerases (Pols), in which one inserts the nucleotide opposite the lesion and the other carries out the subsequent extension. By extending from the nucleotide inserted by another Pol, Polζ plays an indispensable role in mediating lesion bypass. Polζ comprises the Rev3 catalytic and Rev7 accessory subunits. Pol32, a subunit of the replicative polymerase Polδ, is also required for Polζ-dependent TLS, but how this Polδ subunit contributes to Polζ function in TLS has remained unknown. Here we show that yeast Polζ is a four-subunit enzyme containing Rev3, Rev7, Pol31, and Pol32; in this complex, association with Pol31/Pol32 is mediated via binding of the Rev3 C terminus to Pol31. The functional requirement of this complex is supported by evidence that mutational inactivation of Rev3's ability to bind Pol31 abrogates Polζ's role in TLS in yeast cells. These findings identify an unexpected role of Pol31 and Pol32 as two essential subunits of Polζ, and clarify why these proteins are required for Polζ-dependent TLS, but not for TLS mediated by Polη in yeast cells. To distinguish the four-subunit complex from the two-subunit Polζ, we designate the four-subunit enzyme "Polζ-d," where "-d" denotes the Pol31/Pol32 subunits of Polδ.
Asunto(s)
ADN Polimerasa III/inmunología , ADN de Hongos/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/inmunología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN Polimerasa III/genética , ADN de Hongos/genética , ADN Polimerasa Dirigida por ADN/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits.
Asunto(s)
ADN Polimerasa III/fisiología , Antígeno Nuclear de Célula en Proliferación/química , Secuencia de Bases , Western Blotting , ADN Polimerasa III/química , ADN Polimerasa III/inmunología , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase delta efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase delta is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase delta, RFC and PCNA are required for large loop DNA repair synthesis.
Asunto(s)
ADN Polimerasa III/fisiología , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Antígeno Nuclear de Célula en Proliferación/fisiología , Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , ADN Polimerasa III/antagonistas & inhibidores , ADN Polimerasa III/inmunología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/inmunología , Sueros Inmunes/farmacología , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/metabolismo , Antígeno Nuclear de Célula en Proliferación/inmunología , Proteína de Replicación C , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismoRESUMEN
The delta and delta' subunits are essential components of the DNA polymerase III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (tau2gammadeltadelta'chipsi). The x-ray crystal structure of delta' contains three structural domains (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localize the delta-binding domain of delta' to a carboxyl-terminal domain III by quantifying the interaction of delta with a series of delta' fusion proteins lacking specific domains. Purification and immobilization of the fusion proteins were facilitated by the inclusion of a tag containing hexahistidine and a short biotinylation sequence. Both NH2- and COOH-terminal-tagged full-length delta' were soluble and had specific activities comparable with that of native delta'. delta and delta' form a 1:1 heterodimer with a dissociation constant (K(D)) of 5 x 10(-7) m determined by equilibrium sedimentation. The K(D) determined by surface plasmon resonance was comparable. Domain III alone bound delta at an affinity comparable to that of wild type delta', whereas proteins lacking domain III did not bind delta. Using a panel of domain-specific anti-delta' monoclonal antibodies, we found that two of the domain III-specific monoclonal antibodies interfered with delta-delta' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.
Asunto(s)
ADN Polimerasa III/metabolismo , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Cromatografía Liquida , Cristalografía por Rayos X , ADN Polimerasa III/química , ADN Polimerasa III/inmunología , ADN Polimerasa III/aislamiento & purificación , Cartilla de ADN , Replicación del ADN , Electroforesis en Gel de Poliacrilamida , Unión ProteicaRESUMEN
The nuclear-encoded DNA polymerase gamma (DNA POL gamma) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POL gamma and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POL gamma gene from Drosophila melanogaster and a partial cDNA for DNA POL gamma from Gallus gallus have been cloned. The predicted human DNA POL gamma polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those of Schizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster, and the C-terminal half of G. gallus, respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POL gamma like activity from mitochondrial extracts. The human DNA POL gamma is unique in that the first exon of the gene contains a CAG10 trinucleotide repeat.
Asunto(s)
ADN Polimerasa III/genética , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Pollos , Mapeo Cromosómico , Cromosomas Humanos Par 15 , Clonación Molecular , ADN Polimerasa III/inmunología , ADN Polimerasa III/metabolismo , ADN Complementario , Drosophila melanogaster/genética , Exones , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Repeticiones de TrinucleótidosRESUMEN
The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of physical and immunological methods. A highly specific rabbit antiserum directed against the native enzyme was developed and found to recognize specifically its two subunits in immunoblot and immunoprecipitation analyses. That and the potent inhibition by the rabbit antiserum of the DNA polymerase and 3'-->5' exonuclease activities of the nearly homogeneous mitochondrial DNA polymerase provide strong evidence for the physical association of the 3'-->5' exonuclease with the two subunit enzyme. An immunoprecipitation analysis of crude enzyme fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally distinct. Template-primer DNA binding studies demonstrated formation of a stable and discrete enzyme-DNA complex in the absence of accessory proteins. Photochemical cross-linking of the complexes by UV light indicated that the alpha but not the beta subunit of mitochondrial DNA polymerase makes close contact with DNA, and limited digestion of the native enzyme with trypsin showed that an approximately 65-kDa proteolytic fragment of the alpha subunit retains the DNA binding function.
Asunto(s)
ADN Polimerasa III/química , Drosophila/enzimología , Mitocondrias/enzimología , Animales , Secuencia de Bases , ADN Polimerasa III/inmunología , ADN Polimerasa III/metabolismo , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Drosophila/embriología , Sueros Inmunes/inmunología , Larva/enzimología , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Conejos , Moldes GenéticosRESUMEN
Using a cell-free system for UV mutagenesis we have recently shown that extracts prepared from Escherichia coli cells promote a UV mutagenesis pathway that depends on the uvrABC repair genes independent of DNA replication (type II UV mutagenesis; Cohen-Fix, O., and Livneh, Z. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3300-3304). Type II UV mutagenesis was defective also in extracts prepared from a uvrD strain. These deficiencies were complemented by adding purified UvrA, UvrB, UvrC, or UvrD proteins to the respective cell extracts. The Uvr proteins act at an early stage in the process, probably preparing a premutagenic single-stranded DNA gap, which subsequently serves as a substrate for the mutagenic reaction. Type II UV mutagenesis was not dependent on DNA polymerases I or on DNA polymerase II, but it was dependent on DNA polymerase III. Thus, similar to the in vivo situation, only DNA polymerase III is essential for UV mutagenesis. Antibodies against the beta subunit of DNA polymerase III holoenzyme inhibited DNA replication but not UV mutagenesis. Thus, the processivity subunit of the holoenzyme is not required for type II UV mutagenesis, in agreement with a mechanism involving filling-in of short single-stranded DNA gaps.
Asunto(s)
Daño del ADN , ADN Helicasas , Reparación del ADN/genética , ADN Bacteriano/efectos de la radiación , Endodesoxirribonucleasas , Proteínas de Escherichia coli , Escherichia coli/efectos de la radiación , Genes Bacterianos/efectos de la radiación , Mutagénesis , Rayos Ultravioleta , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Anticuerpos/farmacología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Secuencia de Bases , ADN Polimerasa I/metabolismo , ADN Polimerasa I/efectos de la radiación , ADN Polimerasa II/metabolismo , ADN Polimerasa II/efectos de la radiación , ADN Polimerasa III/inmunología , ADN Polimerasa III/metabolismo , ADN Polimerasa III/efectos de la radiación , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , ADN Bacteriano/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Genotipo , Sustancias Macromoleculares , Datos de Secuencia MolecularRESUMEN
Highly purified preparations of chick embryo DNA polymerase gamma contained 3'----5' exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T.A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase gamma precipitated 3'----5' exonuclease activity to the same extent as DNA polymerase gamma activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase gamma activity to thermal treatment at 50 degrees C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase gamma and 3'----5' exonuclease are, although not identical, closely correlated.
Asunto(s)
Anticuerpos/inmunología , ADN Polimerasa III/inmunología , ADN Polimerasa Dirigida por ADN/inmunología , Exodesoxirribonucleasas/inmunología , Animales , Sitios de Unión , Bovinos , Precipitación Química , Embrión de Pollo , ADN Polimerasa III/metabolismo , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/metabolismo , Calor , ConejosRESUMEN
In Escherichia coli the dnaQ+ gene, which encodes epsilon, a fidelity subunit of DNA polymerase III, and the rnh+ gene, which encodes RNase H, share a promoter region but are transcribed in opposite directions. The presence of this divergent transcriptional unit on a multicopy plasmid inhibited by as much as 10-fold mutations induced by the SOS-dependent mutagens methyl methanesulfonate and UV light. Mutations in either gene eliminated the effect, suggesting that both genes contribute either directly or indirectly to the antimutagenic phenotype. Neither survival to mutagen exposure nor induction of the SOS response was comparably affected by the presence of the genes. Although the antimutagenic phenotype was partially suppressed by excess UmuDC proteins, which are required for SOS mutagenesis, the presence of the dnaQ+-rnh+ clone also reduced the induction of mutations by N-methyl-N'-nitro-N-nitrosoguanidine in cells deficient for SOS mutagenic processing. The results suggest that the presence of the dnaQ+-rnh+ divergent transcriptional unit interferes with an underlying mutagenic mechanism that is normally facilitated by the proteins induced as part of the SOS response.
Asunto(s)
ADN Polimerasa III/genética , Reparación del ADN , ADN Polimerasa Dirigida por ADN/genética , Endorribonucleasas/fisiología , Escherichia coli/genética , Mutación , Respuesta SOS en Genética , Western Blotting , Clonación Molecular , ADN Polimerasa III/inmunología , Replicación del ADN , Genes Bacterianos , Genotipo , Metilnitronitrosoguanidina/farmacología , Plásmidos , Rec A Recombinasas/genética , Ribonucleasa H , Supresión Genética , Transcripción GenéticaRESUMEN
Monoclonal antibodies directed against the alpha subunit of the DNA polymerase III holoenzyme (1) of E. coli were tested for cross-reactivity with a variety of polymerases. We found that one monoclonal antibody bound to E. coli DNA polymerase I as well as to DNA polymerase III. A weaker, but specific, interaction was also detected with T4 DNA polymerase. We exploited the proteolysis procedure developed by Setlow, Brutlag and Kornberg (2) to determine which domain of DNA polymerase I contained the conserved epitope. Contrary to expectations, it was not found in the polymerase domain, but in the 5'----3' exonuclease domain. This reveals a sequence or structure, sufficiently important to be conserved among these polymerases, that is not directly involved in the polymerization reaction.
Asunto(s)
ADN Polimerasa III/inmunología , ADN Polimerasa I/inmunología , ADN Polimerasa Dirigida por ADN/inmunología , Anticuerpos Monoclonales/inmunología , Reacciones Cruzadas , ADN/biosíntesis , Epítopos , Técnicas de Inmunoadsorción , Fagos T/enzimologíaRESUMEN
We have established two murine hybridoma cell lines that secrete monoclonal antibodies directed against the tau subunit of the DNA polymerase III holoenzyme of Escherichia coli. Both antibodies have been purified and identified to be of the IgG1 class. Competition assays indicate that they bind to two distinct portions of the tau subunit. These antibodies have been used to demonstrate that tau is an integral part of all DNA polymerase III holoenzyme assemblies and that tau is the product of the dnaZX gene. Both of the antibodies react only with tau, not with gamma, the other protein product of the dnaZX gene. Immunoprecipitation studies demonstrated that tau is contained within the same enzyme assemblies as gamma (dnaZ protein). This observation is discussed in the light of the DNA polymerase III holoenzyme functioning as an asymmetric dimer, capable of coordinating leading with lagging strand replication.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/inmunología , ADN Polimerasa III/inmunología , ADN Polimerasa Dirigida por ADN/inmunología , Escherichia coli/enzimología , Genes Bacterianos , Complejos Multienzimáticos/análisis , Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales/genética , Proteínas Bacterianas/genética , ADN Polimerasa III/genética , Escherichia coli/genética , Escherichia coli/inmunología , GenesRESUMEN
We have established three murine hybridoma cell lines that secrete monoclonal antibodies directed specifically against the alpha subunit of the DNA polymerase III holoenzyme of Escherichia coli. All three antibodies have been purified and identified to be of the IgM class. competition binding assays indicate that these antibodies bind to at least two distinct but adjacent or interacting sites. An immunoblot assay has been developed that permits quantitation of alpha in crude extracts. These assays agree with previous determinations of the number of polymerase molecules present/cell and indicate that the size of alpha is the same in both the crude and pure form. Anti-alpha IgM can be used with fixed Staphylococcus aureus, cells and anti-IgM to specifically precipitate alpha.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , ADN Polimerasa III/inmunología , ADN Polimerasa Dirigida por ADN/inmunología , Escherichia coli/enzimología , Proteínas de Insectos , Receptores de Superficie Celular/análisis , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Peso MolecularRESUMEN
The initiation of the DNA polymerase III holoenzyme-catalyzed reaction is blocked by antibody directed against the beta subunit; elongation is unaffected (Johanson, K., and McHenry, C. (1980) J. Biol. Chem. 255, 10984-10990). We have developed an immunological method for quantitating nanogram quantities of beta in reaction complexes. Using this method, we have demonstrated that beta is present in all stages of the DNA polymerase III holoenzyme reaction. Upon initiation complex formation, the antigenic determinants of beta become inaccessible to anti-beta immunoglobulin G. The methods described herein should be generally applicable to the study of a variety of multienzyme complexes. Even after conversion of a primed G4 single strand to the duplex replicative form, beta does not readily dissociate. This creates a kinetic barrier to the overall holoenzyme replicative reaction.
Asunto(s)
Anticuerpos/inmunología , ADN Polimerasa III/análisis , ADN Polimerasa Dirigida por ADN/análisis , ADN/metabolismo , ADN Polimerasa III/inmunología , Cinética , Sustancias Macromoleculares , Relación Estructura-ActividadRESUMEN
Bacillus subtilis DNA polymerase III (pol III), an arylhydrazinopyrimidine-sensitive, replication-specific enzyme, was used to generate a non-precipitating rabbit antibody which specifically inhibited pol III activity in vitro. The antibody was used to examine structural relationships among several DNA polymerases, and it was linked covalently to agarose; the antibody:agarose was employed to develop a rapid, selective method of purification of catalytically active B. subtilis pol III.
Asunto(s)
Anticuerpos , Bacillus subtilis/enzimología , ADN Polimerasa III/aislamiento & purificación , Replicación del ADN , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Animales , Reacciones Antígeno-Anticuerpo , ADN Polimerasa III/inmunología , Cinética , Peso Molecular , Conejos/inmunología , Especificidad de la EspecieRESUMEN
An antiserum has been prepared against a highly purified DNA polymerase gamma from NC37 cells, a normal human lymphoblast cell line. The antiserum does not possess enzyme neutralizing activity, but does bind specifically to DNA polymerase gamma. When tested in a double antibody immunoprecipitation assay, the antibody does not cross-react with DNA polymerases alpha or beta, purified from NC37 cells, or with reverse transcriptases of avian, murine, or primate RNA tumor viruses. Antisera prepared against purified reverse transcriptases similarly do not recognize DNA polymerase gamma, either in an enzyme neutralization assay or in the more sensitive double antibody immunoprecipitation assay. The availability of an antiserum to DNA polymerase gamma will allow the further characterization of enzyme activities isolated from cellular material and suspected of being related to viral reverse ttranscriptases. In those cases where such activities do not immunologically resemble known viral DNA polymerases, the anti-DNA polymerase gamma will help determine the viral or cellular nature of the unknown activity.
Asunto(s)
ADN Polimerasa III/inmunología , ADN Polimerasa Dirigida por ADN/inmunología , Linfocitos/enzimología , Nucleotidiltransferasas/análisis , Virus Oncogénicos/enzimología , Reacciones Antígeno-Anticuerpo , Línea Celular , Reacciones Cruzadas , ADN Polimerasa II , ADN Polimerasa III/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/análisis , Humanos , Sueros Inmunes , Nucleotidiltransferasas/inmunología , Pruebas de Precipitina , ADN Polimerasa Dirigida por ARN/análisisRESUMEN
We have previously reported [(Ohno, T., Sweet, R.W., Hu, R., DeJak, D. & Spiegelman, S. (1977) Proc. Natl. Acad. Sci. USA 74, 764-768)] on the purification and characterization of the DNA polymerase from human breast cancer particles. Its preference for certain synthetic templates and its ability to use a viral RNA to fashion a faithful DNA transcript identify it as a reverse transcriptase similar to that found in the mouse mammary tumor virus and in the Mason-Pfizer monkey virus (MPMV). We report here that the human breast cancer enzyme crossreacts immunologically with the reverse transcriptase of MPMV. The crossreactivity was shown both by inhibition of enzyme activity and by complex formation between purified enzyme and isolated IgG against MPMV polymerase. No such interactions were observed with other oncornavirus reverse transcriptases of avian, murine, feline, or simian origin. Further, the IgG failed to neutralize the reverse transcriptases from human mesenchymal neoplasias (leukemias and lymphomas) or the activities of normal cellular DNA polymerases (alpha, beta, gamma).