RESUMEN
Cleavage and DNA joining reactions, carried out by human immunodeficiency virus type 1 (HIV-1) integrase, are necessary to effect the covalent insertion of HIV-1 DNA into the host genome. For the integration of HIV-1 DNA into the cellular genome to be completed, short gaps flanking the integrated proviral DNA must be repaired. It has been widely assumed that host cell DNA repair enzymes are involved. Here we report that HIV-1 integrase multimers possess an intrinsic DNA-dependent DNA polymerase activity. The activity was characterized by its dependence on Mg2+, resistance to N-ethylmaleimide, and inhibition by 3'-azido-2',3'-dideoxythymidine-5'-triphosphate, coumermycin A1, and pyridoxal 5'-phosphate. The enzyme efficiently utilized poly(dA)-oligo(dT) or self-annealing oligonucleotides as a template primer but displayed relatively low activity with gapped calf thymus DNA and no activity with poly(dA) or poly(rA)-oligo(dT). A monoclonal antibody binding specifically to an epitope comprised of amino acids 264 to 273 near the C terminus of HIV-1 integrase severely inhibited the DNA polymerase activity. A deletion of 50 amino acids at the C terminus of integrase drastically altered the gel filtration properties of the DNA polymerase, although the level of activity was unaffected by this mutation. The DNA polymerase efficiently extended a hairpin DNA primer up to 19 nucleotides on a T20 DNA template, although addition of the last nucleotide occurred infrequently or not at all. The ability of integrase to repair gaps in DNA was also investigated. We designed a series of gapped molecules containing a single-stranded region flanked by a duplex U5 viral arm on one side and by a duplex nonviral arm on the other side. Molecules varied structurally depending on the size of the gap (one, two, five, or seven nucleotides), their content of T's or C's in the single-stranded region, whether the CA dinucleotide in the viral arm had been replaced with a nonviral sequence, or whether they contained 5' AC dinucleotides as unpaired tails. The results indicated that the integrase DNA polymerase is specifically designed to repair gaps efficiently and completely, regardless of gap size, base composition, or structural features such as the internal CA dinucleotide or unpaired 5'-terminal AC dinucleotides. When the U5 arm of the gapped DNA substrate was removed, leaving a nongapped DNA template-primer, the integrase DNA polymerase failed to repair the last nucleotide in the DNA template effectively. A post-gap repair reaction did depend on the CA dinucleotide. This secondary reaction was highly regulated. Only two nucleotides beyond the gap were synthesized, and these were complementary to and dependent for their synthesis on the CA dinucleotide. We were also able to identify a specific requirement for the C terminus of integrase in the post-gap repair reaction. The results are consistent with a direct role for a heretofore unsuspected DNA polymerase function of HIV-1 integrase in the repair of short gaps flanking proviral DNA integration intermediates that arise during virus infection.
Asunto(s)
Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Integrasa de VIH/metabolismo , VIH-1/enzimología , Animales , Anticuerpos Monoclonales/inmunología , Catálisis , Bovinos , Cartilla de ADN , Didesoxinucleótidos , Integrasa de VIH/genética , Integrasa de VIH/aislamiento & purificación , Humanos , Peso Molecular , Pruebas de Neutralización , Eliminación de Secuencia , Especificidad por Sustrato , Moldes Genéticos , Nucleótidos de Timina/farmacología , Factores de Tiempo , Zidovudina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
The minute virus of mice (MVM) genome is a linear single-stranded length of approximately 5000 nucleotides of DNA with unique terminal palindromic sequences at both ends. The left(3') hairpin is used to prime the initiation of DNA synthesis on parental single-strand DNA while the right (5') hairpin or stem-plus-arms structure can also prime the initiation of DNA synthesis during synthesis of dimer and higher oligomers as well as synthesis of progeny single strands. Previous studies have shown that if viral duplex DNA was input into an in vitro DNA replication system using extracts from uninfected HeLa cells, the 5' end of the molecule was able to form a hairpin and initiate DNA synthesis by DNA polymerase delta (Cossons et al. (1996), Virology 216, 258-264). In this study, the effect of the deletion of known cis-acting genetic elements upon the initiation of DNA replication was studied using a series of MVM mutants with deletions within the 5' terminal region. Mutants containing deletions of elements A (nucleotides 4489-4636), B (nucleotides 4636-4695), and either one or both of the 65-bp repeats (nucleotides 4720-4785 and 4785-4849) were used as template in the in vitro DNA replication system. When element A was deleted, the efficiency of initiation decreased significantly. Subsequent removal of element B, leaving just the two 65-bp repeats, restored levels of initiation back to those seen in the wild-type genome. In the absence of either A or B both 65-bp repeats were necessary for efficient initiation, and removal of one of these repeats caused a decrease in efficiency. Thus, element B appeared to have a negative regulatory effect (in the absence of element A), and element A appeared to have a positive regulatory effect, at least in the presence of element B. These data demonstrate, for the first time, a complex interaction between these cis-acting regulatory elements which can function as both positive or negative regulators in the initiation of MVM DNA replication.
Asunto(s)
Replicación del ADN , Virus Diminuto del Ratón/genética , Secuencias Reguladoras de Ácidos Nucleicos , Replicación Viral , Animales , ADN Viral/biosíntesis , ADN Viral/genética , Células HeLa , Humanos , Ratones , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression in Escherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6xHis tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8-7.2 in the presence of 2 mM MnCl(2). Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3' end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5' oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3' end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5'-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another. Copyright 1996 S. Karger AG, Basel
RESUMEN
The parvovirus the minute virus of mice (MVM) has a linear single-stranded DNA genome with unique palindromic sequences at both termini which enable it to fold back on itself and form hairpin or cruciform-type structures. The purpose of this study was to examine the primary events occurring during MVM replication mediated solely by the host cell replication machinery. In an in vitro DNA replication system using HeLa cell extracts, we found that there was a distinct activity that utilized the 5' terminal palindrome sequence of MVM to produce a secondary structure from a duplex extended form, in a time-dependent fashion. The secondary structure was due to the formation of a hairpin rather than a stem-plus-arms type structure and was associated with initiation of DNA synthesis, performed specifically by DNA polymerase delta. Inhibition of DNA polymerase alpha had no effect upon this activity. Removal of all but 13 base pairs of the hairpin arm abolished the synthesis of DNA, indicating that there is a minimal length requirement for the duplex region of DNA or that this region contains regulatory genetic elements. These data are consistent both with the role of DNA polymerase delta in extending the synthesis of DNA from a DNA primer and with unidirectional continuous DNA synthesis, initiating from a hairpin, as a mode of replication for MVM.
Asunto(s)
ADN Viral/química , ADN Polimerasa Dirigida por ADN/metabolismo , Virus Diminuto del Ratón/genética , Conformación de Ácido Nucleico , Animales , Secuencia de Bases , ADN Polimerasa III , ADN Viral/biosíntesis , ADN Viral/metabolismo , Genoma Viral , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
We have attempted to relate genetic recombination involving human immunodeficiency virus type 1 (HIV-1) to multiple drug resistance by using PEG to fuse subclones of U937 cells that carried HIV-1 recombinants resistant to either 3' -azido-3'-deoxythymidine (AZT) or the (--) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). The parental viruses employed contained well-defined mutations in the pol gene. Fused cells were co-cultured with the MT4 lymphocyte cell line for virus amplification to yield progeny that, in some cases, possessed different patterns of drug resistance from parental viruses. Mutational analyses were performed by PCR to substantiate these observations, which were also confirmed by direct sequencing of single strands of DNA segments, obtained from plaque-purified viruses. These studies indicate that viral recombination had occurred, and establish a theoretical basis on which to conclude that the acquisition of multiple drug resistance on the part of HIV-1 may be related to its ability to promote cell fusion.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Recombinación Genética , Zalcitabina/análogos & derivados , Zidovudina/farmacología , Secuencia de Bases , Fusión Celular , Línea Celular , Técnicas de Cocultivo , Análisis Mutacional de ADN , Resistencia a Múltiples Medicamentos/genética , Genes pol/genética , VIH-1/genética , Humanos , Lamivudine , Datos de Secuencia Molecular , Monocitos , Reacción en Cadena de la Polimerasa/métodos , Linfocitos T , Cultivo de Virus , Zalcitabina/farmacologíaRESUMEN
A mammalian expression vector designed for production of HIV-1 integrase was found to enhance the stability of a linear reporter plasmid in COS-7 cells. The effect is strictly dependent on coexpression of the HIV-1 rev gene and on the inclusion of U3 and U5 portions of the HIV-1 LTR in the reporter plasmid. Integrase point mutations P109S and D116N drastically reduced stabilization whereas T115A and D64A had little or no effect. Immunoblot analysis revealed the presence of a 32-34kDa integrase protein in extracts of transfected COS-7 cells and of wild type and mutant integrase proteins at comparable levels. We conclude that integrase acts in trans in COS-7 cells, possibly by binding to the HIV-1 LTR in the plasmid. This transfection system may be useful for studying factors that stabilize the HIV-1 DNA genome prior to its integration into the host cell chromosome.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Genes rev , Duplicado del Terminal Largo de VIH , VIH-1/enzimología , VIH-1/genética , Plásmidos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , ADN Nucleotidiltransferasas/análisis , ADN Nucleotidiltransferasas/biosíntesis , Humanos , Integrasas , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Mutación Puntual , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Transfección , Integración ViralAsunto(s)
Síndromes de Inmunodeficiencia/genética , Ratones Transgénicos/genética , Proteínas Tirosina Quinasas/genética , Agammaglobulinemia Tirosina Quinasa , Animales , Ensayo de Unidades Formadoras de Colonias , ADN Complementario/genética , Elementos de Facilitación Genéticos , Femenino , Genes Dominantes , Terapia Genética , Hematopoyesis , Cadenas Pesadas de Inmunoglobulina/genética , Síndromes de Inmunodeficiencia/terapia , Tejido Linfoide/patología , Masculino , Ratones , Ratones Mutantes , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Linfocitos T/enzimologíaRESUMEN
DNA polymerase alpha-primase complexes in extracts of MVM-infected murine cells were dissociated in the presence of 0.3M KCl to generate a 12S DNA primase and a 10S DNA polymerase alpha that were readily separated by sedimentation in glycerol gradients. A 12S DNA polymerase alpha-primase complex refractory to dissociation in 0.3M KCl was identified in extracts of MVM-infected HeLa cells. In extracts of mock-infected murine and HeLa cells DNA primase and DNA polymerase alpha were not dissociated from each other in 0.3M KCl but remained in a stable complex that sedimented at 10S. We propose that a novel 12S DNA polymerase alpha-primase complex prone to disruption by salt is induced by MVM infection and that the DNA primase component of the complex is modified.
Asunto(s)
Carcinoma de Ehrlich/enzimología , ADN Polimerasa II/aislamiento & purificación , Virus Diminuto del Ratón/enzimología , ARN Nucleotidiltransferasas/aislamiento & purificación , ARN Nucleotidiltransferasas/metabolismo , Animales , Línea Celular Transformada , Transformación Celular Viral/fisiología , Células Cultivadas , Cricetinae , Cricetulus , ADN Primasa , ADN Viral/biosíntesis , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Sales (Química)RESUMEN
The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5' ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5' terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.
Asunto(s)
Virus Diminuto del Ratón/genética , Tirosina/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , Replicación del ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenilalanina/genética , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Activación Transcripcional , TransfecciónRESUMEN
Long-term bone marrow cultures have been useful in determining gene expression patterns in pre-B cells and in the identification of cytokines such as interleukin 7 (IL-7). We have developed a culture system to selectively grow populations of B lineage restricted progenitors (pro-B cells) from murine bone marrow. Pro-B cells do not grow in response to IL-7, Steel locus factor (SLF), or a combination of the two. c-kit, the SLF receptor, and the IL-7 receptor are both expressed by pro-B cells, indicating that the lack of response is not simply due to the absence of receptors. Furthermore, SLF is not necessary for the growth of pro-B cells since they could be expanded on a stromal line derived from Steel mice that produces no SLF. IL-7 responsiveness in pre-B cells is associated with an increase in n-myc expression and is correlated with immunoglobulin (Ig) gene rearrangements. Although members of the ets family of transcription factors and the Pim-1 kinase are expressed by pro-B cells, n-myc is not expressed. Pro-B cells maintain Ig genes in the germline configuration, which is correlated with a low level of recombination activating genes 1 and 2 (Rag-1 and 2) mRNA expression, but high expression of sterile mu and terminal deoxynucleotidyl transferase. Pro-B cells are unable to grow separated from the stromal layer by a porous membrane, indicating that stromal contact is required for growth. These results suggest that pro-B cells are dependent on alternative growth signals derived from bone marrow stroma and can be distinguished from pre-B cells by specific patterns of gene expression.
Asunto(s)
Linfocitos B/citología , Animales , Secuencia de Bases , Northern Blotting , Células de la Médula Ósea , Diferenciación Celular , División Celular , Supervivencia Celular , Células Cultivadas , ADN , Genes de Inmunoglobulinas , Células Madre Hematopoyéticas/citología , Interleucina-7/fisiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-kit , Receptores Inmunológicos/metabolismo , Receptores de Interleucina-7RESUMEN
The NS-1 gene of minute virus of mice encodes a multifunctional protein required for replication of the viral genome and for transcriptional regulation of the two MVM promoters. To study the localization of activities required for DNA replication and transactivation of the capsid gene promoter, insertion and point mutations were introduced into the NS-1 gene. The mutant NS-1 genes were expressed in COS-7 cells by using an SV 40 promoter driven NS-1 expression vector. The ability of the mutant proteins to complement a replication defective NS-1 mutant of the infectious MVM plasmid pMM984 and to activate transcription from the capsid gene promoter in chloramphenicol acetyl transferase expression assays was determined. Two point mutations Ser-249 to Ala and Lys-250 to Gln and a one amino acid insertion between Asp-606 and Leu-607 had no effect on viral DNA replication and transactivation activities. Six independent insertions of between 2 and 12 amino acids inhibited the DNA replication activity of NS-1 between 20- and at least 100-fold. There was no apparent correlation between the extent of inhibition of parvoviral DNA replication and the location of the mutations. The transcriptional activation function of NS-1 was inhibited between 1.5- and at least 20-fold and was therefore overall relatively less sensitive to mutagenesis than was its DNA replication function. An exception to this was a 5 amino acid insertion between Tyr-543 and Gln-544 that abolished transactivation as well as the ability of NS-1 to complement viral DNA replication.
Asunto(s)
Cápside/genética , Replicación del ADN/genética , Virus Diminuto del Ratón/genética , Mutación , Transactivadores/genética , Proteínas del Núcleo Viral/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Clonación Molecular , ADN Viral/biosíntesis , Regulación Viral de la Expresión Génica , Prueba de Complementación Genética , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Fenotipo , Regiones Promotoras Genéticas , Mapeo Restrictivo , Transcripción Genética , Transfección , Proteínas no Estructurales ViralesRESUMEN
Mutations were introduced into plasmid pMM984, a full-length infectious clone of the fibrotropic strain of minute virus of mice, to identify cis-acting genetic elements required for the excision and replication of the viral genome. The replicative capacity of these mutants was measured directly, using an in vivo transient DNA replication assay following transfection of plasmids into murine A9 cells and primate COS-7 cells. Experiments with subgenomic constructs indicated that both viral termini must be present on the same DNA molecule for replication to occur and that the viral nonstructural protein NS-1 must be provided in trans. The necessary sequences were located within 1,084 and 807 nucleotides of the 3' and 5' ends of the minute virus of mice genome, respectively. The inhibitory effect of deletions within the 206-bp 5'-terminal palindrome demonstrated that these sequences comprise a cis-acting genetic element that is absolutely essential for the excision and replication of viral DNA. The results further indicated a requirement for a stem-plus-arms T structure as well as for the formation of a simple hairpin. In addition, the removal of one copy of a tandemly arranged 65-bp repeat found 94 nucleotides inboard of the 5'-terminal palindrome inhibited viral DNA replication in cis by 10- and just greater than 100-fold in A9 and COS-7 cells, respectively. The latter results define a novel genetic element within the 65-bp repeated sequence, distinct from the terminal palindrome, that is capable of regulating minute virus of mice DNA replication in a species-specific manner.
Asunto(s)
Replicación del ADN , ADN Viral/genética , Genes Virales , Virus Diminuto del Ratón/genética , Animales , Secuencia de Bases , Línea Celular , Deleción Cromosómica , Prueba de Complementación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Insercional , Hibridación de Ácido Nucleico , Plásmidos , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , TransfecciónRESUMEN
We find that the purified murine DNA polymerase.alpha-primase complex exhibits the greatest affinity for DNA templates in which CCC occurs 10 nucleotides downstream of a DNA primase initiation site (Km = 6.6 +/- 0.3 pM). Templates with 3'-CCA-5' at this position are less efficiently utilized (Km = 16 +/- 4 pM). Point mutations that disrupt the 3'-CC(C/A)-5' motif further decrease the affinity for DNA approximately 7-fold (Km = 105 +/- 58 pM). Mutations at the primase start site reduce Vmax 2-fold. Template pyrimidines are required for priming, and initiation with ATP is preferred to initiation with GTP. We conclude that a component of the DNA polymerase.alpha-primase complex recognizes a 3'-CC(C/A)-5' motif in the DNA template, downstream of a primase start site, and that this interaction controls site selection and frequency of initiation by DNA primase.
Asunto(s)
Replicación del ADN , ADN Viral/genética , ARN Nucleotidiltransferasas/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Primasa , Genes Virales , Virus Diminuto del Ratón/genética , Datos de Secuencia Molecular , Mutación , Mapeo Nucleótido , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , Mapeo Restrictivo , Moldes GenéticosRESUMEN
DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.
Asunto(s)
ADN Polimerasa II/metabolismo , Replicación del ADN , ADN/biosíntesis , Escherichia coli/genética , Animales , Secuencia de Bases , Colifagos/genética , ADN Polimerasa I/metabolismo , ADN Recombinante/biosíntesis , ADN de Cadena Simple/aislamiento & purificación , ADN Viral/aislamiento & purificación , Cinética , Ratones , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Mapeo Restrictivo , TransfecciónRESUMEN
Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.
Asunto(s)
Carcinoma de Ehrlich/enzimología , ADN Polimerasa II/aislamiento & purificación , Infecciones por Parvoviridae/enzimología , ARN Nucleotidiltransferasas/análisis , Animales , Afidicolina , Carcinoma de Ehrlich/microbiología , Línea Celular , Cromatografía por Intercambio Iónico/métodos , ADN Polimerasa II/genética , ADN Primasa , ADN Viral/metabolismo , Diterpenos/farmacología , Inducción Enzimática/efectos de los fármacos , Ratones , Virus Diminuto del Ratón/genética , Mutación , Infecciones por Parvoviridae/genética , Replicación ViralRESUMEN
Virions of minute virus of mice were purified by sedimentation in sucrose gradients and chromatography on DEAE-cellulose columns and shown to consist of single-stranded viral DNA and the viral capsid polypeptides VP-1 (83 kDa) and VP-2 (64.5 kDa). A 63-kDa polypeptide distinct from the viral capsid polypeptide VP-3 (61.4 kDa) was found in some virion preparations. Virions sedimented at 135 and 110 S. The genomic single strands associated with purified 135 and 110 S virions were covalently bound to a protein as judged by the anomalous electrophoretic mobility of the DNA in agarose gels at pH 12.5. The protein was removed from the DNA by Pronase but remained bound after heating at 98 degrees in the presence of 0.1% sodium dodecyl sulfate. Nuclease digestion of the purified DNA-protein complex released several polypeptides ranging in size from 58 to 65 kDa. Restriction enzyme analysis of the purified DNA protein complex following its conversion to a duplex RF DNA in vitro showed that the protein was attached to the 5' termini of the DNA.
Asunto(s)
ADN de Cadena Simple/análisis , ADN Viral/análisis , Virus Diminuto del Ratón/análisis , Parvoviridae/análisis , Proteínas Virales/análisis , Virión/análisis , Animales , Cápside/análisis , Carcinoma de Ehrlich , Centrifugación por Gradiente de Densidad , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Ratones , Virus Diminuto del Ratón/genética , Péptidos/análisis , Células Tumorales Cultivadas , Virión/genéticaRESUMEN
The mechanism of nonhomologous recombination in murine cells infected with the parvovirus minute virus of mice (MVM) has been investigated by analysis of DNA sequences at recombination junctions in naturally occurring deletion variants of the virus. We report here that nonhomologous recombination in the MVM chromosome is characterized by short homologies, by insertion at recombination junctions of foreign DNA sequences that are enriched for preferred eucaryotic topoisomerase I cleavage sites, and by an association with a common DNA sequence motif of the type 5'-CTATTTCT-3'. Additional analyses of broken MVM chromosomes provided evidence for specific enzymatic cleavage within 5'-CTTATC-3' and 5'-CTATTC-3' sequences. The results indicate that the 5'-CTATTTCT-3' motif is an important genetic element for nonhomologous recombination in the parvovirus chromosome.
Asunto(s)
Cromosomas , Virus Diminuto del Ratón/genética , Parvoviridae/genética , Recombinación Genética , Animales , Secuencia de Bases , Deleción Cromosómica , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Viral/análisis , RatonesRESUMEN
The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.
Asunto(s)
ADN Polimerasa II/genética , Replicación del ADN , ADN Viral/biosíntesis , ARN Nucleotidiltransferasas/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Polimerasa II/análisis , ADN Primasa , ADN Viral/genética , Genes Reguladores , Sustancias Macromoleculares , Ratones , Peso Molecular , ARN Nucleotidiltransferasas/análisis , ARN Viral/biosíntesis , Moldes GenéticosRESUMEN
Previous work (E. A. Faust and D. C. Ward, J. Virol. 32:276-292, 1979) revealed a remarkably high rate of spontaneous deletion in viral DNA during lytic infection of cultured murine cells with minute virus of mice (MVM), an autonomous parvovirus. In the present study, we have isolated plasmid and phage recombinants containing MVM DNA inserts bearing deletions and we have determined the DNA sequence spanning three deletion junctions. The deletions, which average 3 kilobases in length, occur between pairs of perfectly homologous 4- to 10-base-pair direct repeats, such that one copy of the repeated sequence is lost, whereas the other remains behind at the deletion junction. When compared, the three sets of direct repeats exhibit no apparent sequence homology and have an A + T content of between 50 and 80%. These results indicate that 4- to 10-base-pair homologies mediate spontaneous deletion formation in the MVM genome and highlight parvoviruses as novel model systems for studies of this ubiquitous pathway of genetic variation.