RESUMEN
Full-length cDNAs for DNA ligase IV and the alpha and beta isoforms of DNA ligase III were cloned from Xenopus laevis to permit study of the genes encoding mitochondrial DNA ligase. DNA ligase III alpha and III beta share a common NH(2) terminus that encodes a mitochondrial localization signal capable of targeting green fluorescent protein to mitochondria while the NH(2) terminus of DNA ligase IV does not. Reverse transcriptase-polymerase chain reaction analyses with adult frog tissues demonstrate that while DNA ligase III alpha and DNA ligase IV are ubiquitously expressed, DNA ligase III beta expression is restricted to testis and ovary. Mitochondrial lysates from X. laevis oocytes contain both DNA ligase III alpha and III beta but no detectable DNA ligase IV. Gel filtration, sedimentation, native gel electrophoresis, and in vitro cross-linking experiments demonstrate that mtDNA ligase III alpha exists as a high molecular weight complex. We discuss the possibility that DNA ligase III alpha exists in mitochondria in association with novel mitochondrial protein partners or as a homodimer.
Asunto(s)
ADN Ligasas/genética , Mitocondrias/enzimología , Oocitos/enzimología , Xenopus laevis/metabolismo , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular , Clonación Molecular , Reactivos de Enlaces Cruzados/química , ADN Ligasa (ATP) , ADN Ligasas/química , ADN Ligasas/clasificación , ADN Ligasas/metabolismo , Femenino , Genes Reporteros , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Mitocondrias/fisiología , Datos de Secuencia Molecular , Peso Molecular , Oocitos/química , Ovario/enzimología , Filogenia , Proteínas de Unión a Poli-ADP-Ribosa , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Testículo/enzimología , Distribución Tisular , Proteínas de Xenopus , Xenopus laevis/genéticaRESUMEN
A number of laboratories have shown that those types of DNA damage that are generally reparable by base excision repair are efficiently repaired in mtDNA. In contrast, most types of damage that require other sorts of repair machinery are not effectively repaired in mtDNA. We have shown that a set of highly purified mitochondrial proteins, including AP endonuclease (APE), DNA polymerase gamma, and mtDNA ligase, is capable of efficiently repairing abasic (AP) sites in mtDNA. These three enzymes appear to conduct all four steps in a conventional BER mechanism: incision, removal of the 5'-deoxyribosephosphate by dRP lyase, polymerization, and ligation. Both DNA polymerase gamma and mtDNA ligase possess some dRP lyase activity. DNA polymerase gamma is a member of the family A of DNA polymerases, with clear homology to DNA pol I of E. coli, while mtDNA ligase is an alternatively expressed form of DNA ligase III. The dRP lyase activities discovered in these mitochondrial enzymes are not unique, but are found in all representatives tested of the family-A DNA polymerases and of the ATP-dependent DNA ligases. These dRP lyase activities have low turnover rates that may have important implications for the overall process of BER. All proteins involved in maintenance of mtDNA are encoded in the nuclear genome and must be directed to mitochondria in order to act on mtDNA. Thus, it is evident that the scope of DNA repair activities undertaken within mitochondria is determined by the set of nucleus-encoded DNA repair enzymes that are capable of being imported into the organelle. A review of DNA repair proteins that may be imported into mitochondria in various organisms will be presented.
Asunto(s)
ADN Ligasas/genética , Reparación del ADN , ADN Mitocondrial/genética , Animales , Proteínas Bacterianas/genética , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/metabolismo , Núcleo Celular/enzimología , Daño del ADN , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , ADN Polimerasa gamma , ADN Mitocondrial/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleasa IV (Fago T4-Inducido) , Proteínas Fúngicas/genética , Genes , Humanos , Ratones , Estrés Oxidativo , Proteínas de Unión a Poli-ADP-Ribosa , Homología de Secuencia de Aminoácido , Proteínas de Xenopus , Xenopus laevis/metabolismoRESUMEN
Mature Xenopus oocytes are highly enriched for mitochondria. The organelles are stored and partitioned to newly-arising cells during embryogenesis, when there is little mitochondrial DNA (mtDNA) replication or transcription. A previously described member of the high mobility group (HMG) family of proteins, mtTFA, has been suggested to play a role in control of mtDNA copy number. mtTFA serves as a mitochondrial transcription factor in humans and Xenopus and as an abundant mtDNA packaging protein in yeast, like its prokaryotic histone-like counterpart, HU protein. Northern blot analysis demonstrated that expression of the gene was regulated during Xenopus oogenesis and specifically peaked at stage II. Western and Southern blotting were used to quantify amounts of the protein and mtDNA, respectively, in each stage of oogenesis. mtTFA:mtDNA ratios were found to be relatively low in previtellogenic oocytes while the ratios increased markedly in mature oocytes.
Asunto(s)
ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Mitocondriales , Proteínas Nucleares/metabolismo , Oogénesis/genética , Factores de Transcripción/metabolismo , Xenopus laevis , Animales , Southern Blotting , Western Blotting , ADN Mitocondrial/biosíntesis , ADN Mitocondrial/genética , Femenino , Proteínas del Grupo de Alta Movilidad/genética , Proteínas Nucleares/genética , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Xenopus laevis/genética , Xenopus laevis/fisiologíaRESUMEN
Polymerase gamma, which replicates and repairs mitochondrial DNA, requires the Pol gamma B subunit for processivity. We determined the crystal structure of mouse Pol gamma B, a core component of the mitochondrial replication machinery. Pol gamma B shows high similarity to glycyl-tRNA synthetase and dimerizes through an unusual intermolecular four-helix bundle. A human Pol gamma B mutant lacking the four-helix bundle failed to dimerize in solution or to stimulate the catalytic subunit Pol gamma A, but retained the ability to bind with Pol gamma A to a primer-template construct, indicating that the functional holoenzyme contains two Pol gamma B molecules. Other mutants retained stimulatory activity but lost the ability to bind folded ssDNA. These results suggest that the Pol gamma B dimer contains distinct sites for Pol gamma A binding, dimerization, and DNA binding.
Asunto(s)
Replicación del ADN/fisiología , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Eliminación de Gen , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Cristalografía , ADN Polimerasa gamma , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Dimerización , Glicina-ARNt Ligasa/química , Glicina-ARNt Ligasa/genética , Humanos , Mamíferos , Ratones , Mitocondrias/genética , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Propiedades de Superficie , Agua/químicaRESUMEN
Mitochondrial DNA polymerase gamma (pol gamma) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5' side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol gamma acts to catalyze the removal of a 5'-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol gamma is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH(4). For both DNA pol gamma and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol beta, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Proteínas de Escherichia coli , Secuencia de Bases , Catálisis , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , ADN Polimerasa I/metabolismo , ADN Polimerasa gamma , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Mitocondrias/enzimología , Modelos Químicos , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Temperatura , Factores de TiempoRESUMEN
Previous studies have shown that the small subunit of Xenopus DNA polymerase gamma (pol gammaB) acts as a processivity factor to stimulate the 140 kDa catalytic subunit of human DNA polymerase gamma. A putative human pol gammaB initially identified by analysis of DNA sequence had not been shown to be functional, and appeared to be an incomplete clone. In this paper, we report the cloning of full-length human and mouse pol gammaB. Both human and mouse pol gammaB proteins were expressed in their mature forms, without their apparent mitochondrial localization signals, and shown to stimulate processivity of the recombinant catalytic subunit of human pol gammaA. Deletion analysis of human pol gammaB indicated that blocks of sequence conserved with prokaryotic class II aminoacyl-tRNA synthetases are necessary for activity and inter-action with human pol gammaA. Purification of DNA pol gamma from HeLa cells indicated that both proteins are associated in vivo.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/genética , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/metabolismo , Animales , ADN Polimerasa gamma , ADN Mitocondrial/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Peptide sequences obtained from the accessory subunit of Xenopus laevis mitochondrial DNA (mtDNA) polymerase gamma (pol gamma) were used to clone the cDNA encoding this protein. Amino-terminal sequencing of the mitochondrial protein indicated the presence of a 44-amino-acid mitochondrial targeting sequence, leaving a predicted mature protein with 419 amino acids and a molecular mass of 47.3 kDa. This protein is associated with the larger, catalytic subunit in preparations of active mtDNA polymerase. The small subunit exhibits homology to its human, mouse, and Drosophila counterparts. Interestingly, significant homology to glycyl-tRNA synthetases from prokaryotic organisms reveals a likely evolutionary relationship. Since attempts to produce an enzymatically active recombinant catalytic subunit of Xenopus DNA pol gamma have not been successful, we tested the effects of adding the small subunit of the Xenopus enzyme to the catalytic subunit of human DNA pol gamma purified from baculovirus-infected insect cells. These experiments provide the first functional evidence that the small subunit of DNA pol gamma stimulates processive DNA synthesis by the human catalytic subunit under physiological salt conditions.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , ADN Mitocondrial/química , ADN Polimerasa Dirigida por ADN/metabolismo , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Polimerasa gamma , ADN Polimerasa Dirigida por ADN/química , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Cloruro de Potasio/farmacología , Pruebas de Precipitina , Homología de Secuencia de AminoácidoAsunto(s)
Daño del ADN/genética , Reparación del ADN/genética , ADN Mitocondrial/genética , Animales , Liasas de Carbono-Oxígeno/análisis , ADN Ligasas/análisis , ADN Ligasas/fisiología , ADN Polimerasa gamma , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/efectos de la radiación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/fisiología , Desoxirribonucleasa IV (Fago T4-Inducido) , Humanos , VertebradosRESUMEN
Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5'-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5'-dRP group may be removed by AP lyase enzymes that employ a beta-elimination mechanism. This beta-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5'-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA.
Asunto(s)
ADN Ligasas/metabolismo , Reparación del ADN , ADN/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , ADN/genética , Femenino , Datos de Secuencia Molecular , Especificidad por Sustrato , XenopusRESUMEN
Mutations in mitochondrial DNA (mtDNA) cause a variety of relatively rare human diseases and may contribute to the pathogenesis of other, more common degenerative diseases. This stimulates interest in the capacity of mitochondria to repair damage to mtDNA. Several recent studies have shown that some types of damage to mtDNA may be repaired, particularly if the lesions can be processed through a base excision mechanism that employs an abasic site as a common intermediate. In this paper, we demonstrate that a combination of enzymes purified from Xenopus laevis mitochondria efficiently repairs abasic sites in DNA. This repair pathway employs a mitochondrial class II apurinic/apyrimidinic (AP) endonuclease to cleave the DNA backbone on the 5' side of an abasic site. A deoxyribophosphodiesterase acts to remove the 5' sugar-phosphate residue left by AP endonuclease. mtDNA polymerase gamma fills the resulting 1-nucleotide gap. The remaining nick is sealed by an mtDNA ligase. We report the first extensive purification of mtDNA ligase as a 100-kDa enzyme that functions with an enzyme-adenylate intermediate and is capable of ligating oligo(dT) strands annealed to poly(rA). These properties together with preliminary immunological evidence suggest that mtDNA may be related to nuclear DNA ligase III.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , ADN Ligasas/metabolismo , Reparación del ADN , ADN Mitocondrial , ADN Polimerasa Dirigida por ADN/metabolismo , Mitocondrias/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Secuencia de Aminoácidos , Animales , Liasas de Carbono-Oxígeno/clasificación , Fraccionamiento Celular , ADN/biosíntesis , ADN Polimerasa gamma , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Células HeLa , Humanos , Datos de Secuencia Molecular , Moldes Genéticos , Xenopus laevisRESUMEN
The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided evidence of cross-linked dimers, trimers and tetramers of xl-mtTFA. Finally, high resolution scanning transmission electron microscopy (STEM) established that xl-mtTFA binds to the specific promoter-proximal site predominantly as a tetramer. Computer analysis of several previously characterized binding sites for xl-mtTFA revealed a fine structure consisting of two half-sites in a symmetrical orientation. The predominant sequence of this dyad symmetry motif shows homology to binding sites of sequence-specific HMG-box-containing proteins such as Sry and Lef-1. We suggest that bidirectional activation of transcription results from the fact that binding of a tetramer of xl-mtTFA permits symmetrical interactions with other components of the transcription machinery at the adjacent core promoters.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Mitocondrias/genética , Transactivadores/metabolismo , Activación Transcripcional , Proteínas de Xenopus , Animales , Sitios de Unión , Secuencia de Consenso , Reactivos de Enlaces Cruzados , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/ultraestructura , Glutaral/farmacología , Proteínas del Grupo de Alta Movilidad/efectos de los fármacos , Proteínas del Grupo de Alta Movilidad/ultraestructura , Microscopía Electrónica de Rastreo , Nucleoproteínas/ultraestructura , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Transactivadores/efectos de los fármacos , Transactivadores/ultraestructura , Xenopus laevisRESUMEN
Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.
Asunto(s)
ADN Polimerasa III/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Composición de Base , ADN de Cadena Simple , Concentración de Iones de Hidrógeno , Conformación de Ácido Nucleico , Moldes GenéticosRESUMEN
Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells respectively of the African frog Xenopus laevis. In this paper, cDNAs encoding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expressed from a portion of the coding sequence was recognized by an antiserum directed against the somatic topoisomerase I that had previously been shown to be unable to cross-react with the oocyte enzyme. Thus, the clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the active site tyrosine of the somatic topoisomerase I reacts with the enzymes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridization showed that oocytes contain an abundant store of somatic topoisomerase I mRNA that is not efficiently polyadenylated in oocytes. This stored RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in oocytes. Microinjection into oocytes of in vitro transcribed mRNA prepared from a Myc-tagged construct of the somatic topoisomerase I sequence is translated to yield a 110 kDa product. This suggests that the oocyte-specific 165 kDa topoisomerase I is not produced by tissue-specific post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocytes that has not yet been identified.
Asunto(s)
ADN-Topoisomerasas de Tipo I/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Cartilla de ADN/metabolismo , ADN Complementario/química , Drosophila melanogaster , Epítopos , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Peso Molecular , Oocitos/enzimología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Schizosaccharomyces , Xenopus laevisRESUMEN
Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.
Asunto(s)
ADN Polimerasa III/metabolismo , ADN Mitocondrial/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Polimerasa III/genética , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , ADN Mitocondrial/genética , ADN de Cadena Simple/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Femenino , Técnicas In Vitro , Datos de Secuencia Molecular , Proteínas Virales/metabolismo , Xenopus laevisRESUMEN
Transcription of Xenopus laevis mitochondrial DNA requires mtRNA polymerase and a dissociable factor, xl-mtTFB, that is distinct from the HMG-box factor known as mtTFA. This paper presents the purification of mtTFB and characterizes its DNA binding properties. xl-mtTFB activity copurifies with a 40-kDa polypeptide on silver-stained protein gels. Activity can be recovered following elution of this 40-kDa polypeptide from an SDS-polyacrylamide gel. xl-mtTFB is capable of binding to DNA, but this binding is relatively nonspecific and is easily competed by heterologous DNA.
Asunto(s)
ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Femenino , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , Xenopus laevisRESUMEN
We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases.
Asunto(s)
ADN Polimerasa III/genética , Regulación del Desarrollo de la Expresión Génica , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Polimerasa III/química , ADN Polimerasa III/metabolismo , ADN Complementario , ADN Mitocondrial , Femenino , Kluyveromyces/genética , Mitocondrias/genética , Datos de Secuencia Molecular , Oocitos/citología , ARN Mensajero/metabolismo , Conejos , Saccharomycetales/enzimología , Saccharomycetales/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Transcription of mtDNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mtRNA polymerase acting with a 40-kDa dissociable factor known as mtTFB or Mtflp. mtTFB has been cloned and characterized in S. cerevisiae, but has not been studied in similar detail in any other organism. Although it is known that mitochondrial transcription in the dairy yeast, Kluyveromyces lactis, initiates within the same consensus promoter sequence used in S. cerevisiae, no previous studies have focused on the proteins involved in transcription initiation in K. lactis. In this article, we report the cloning of mtTFB from K. lactis and from a yeast more closely related to S. cerevisiae, S. kluyveri. Both novel mtTFB genes were able to substitute for the MTF1 gene in S. cerevisiae. Both proteins purified following expression in E. coli were able to support specific transcription initiation in vitro with the S. cerevisiae mtRNA polymerase. The S. kluyveri and K. lactis mtTFB proteins share only 56% and 40% identity with S. cerevisiae mtTFB, respectively. Alignments of the three mtTFB sequences did not reveal any regions larger than 30 amino acids with greater than 60% amino acid identity. In particular, regions proposed to show sequence similarity to bacterial sigma factors were not more highly conserved than other regions of the mtTFB proteins. All three yeast mtTFB genes lack conventional amino-terminal mitochondrial targeting sequences, suggesting that all three proteins may be imported into mitochondria by the same unusual mechanism reported for S. cerevisiae mtTFB.
Asunto(s)
ADN de Hongos/metabolismo , ADN Mitocondrial/metabolismo , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , ADN de Hongos/química , ADN Mitocondrial/química , Evolución Molecular , Proteínas Fúngicas/genética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas Mitocondriales , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.
Asunto(s)
ADN Mitocondrial/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Mitocondrias/enzimología , Transactivadores , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Femenino , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Moldes Genéticos , Factores de Transcripción/biosíntesis , Factores de Transcripción/química , Xenopus laevisRESUMEN
Mitochondrial DNA is subject to oxidative damage generating 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) residues and to spontaneous or induced base loss generating abasic sites. Synthetic oligonucleotides containing these lesions were prepared and used as templates to determine their effects on the action of Xenopus laevis DNA polymerase gamma. An analogue of an abasic site in DNA, tetrahydrofuran, was found to inhibit elongation by DNA polymerase gamma. When the DNA polymerase was able to complete translesional synthesis, a dA residue was incorporated opposite the abasic site. In contrast, elongation by DNA polymerase gamma was not inhibited by an 8-oxo-dG residue in the template strand. The polymerase inserted dA opposite 8-oxo-dG in approximately 27% of the extended products. The effects of these lesions on the 3'-->5' exonuclease proofreading activity of DNA polymerase gamma were also investigated. The 3'-->5' exonuclease activity excised any of the four normal bases positioned opposite either a tetrahydrofuran residue or 8-oxo-dG, suggesting that proofreading may not play a major role in avoiding misincorporation at abasic sites or 8-oxo-dG residues in the template. Thus, both of these lesions have the prospect of causing high rates of mutation during mtDNA replication.
Asunto(s)
Daño del ADN , ADN Polimerasa III/fisiología , ADN Mitocondrial/metabolismo , Mitocondrias/enzimología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Secuencia de Bases , Replicación del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/farmacología , Femenino , Datos de Secuencia Molecular , Xenopus laevisRESUMEN
DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.