RESUMEN
Sterile alpha motif and histidine/aspartic acid domaincontaining protein 1 (SAMHD1), the only deoxynucleotide triphosphate (dNTP) hydrolase in eukaryotes, plays a crucial role in regulating the dynamic balance and ratio of cellular dNTP pools. Furthermore, SAMHD1 has been reported to be involved in the pathological process of several diseases. Homozygous SAMHD1 mutations have been identified in immune system disorders, such as autoimmune disease AicardiGoutières syndrome (AGS), whose primary pathogenesis is associated with the abnormal accumulation and disproportion of dNTPs. SAMHD1 is also considered to be an intrinsic virusrestriction factor by suppressing the viral infection process, including reverse transcription, replication, packaging and transmission. In addition, SAMHD1 has been shown to promote genome integrity during homologous recombination following DNA damage, thus being considered a promising candidate for oncotherapy applications. The present review summarizes the molecular mechanisms of SAMHD1 regarding the regulation of dNTP homeostasis and DNA damage response. Additionally, its potential effects on tumorigenesis and oncotherapy are reported.
Asunto(s)
Reparación del ADN , Inestabilidad Genómica , Homeostasis , Mutación , Neoplasias/tratamiento farmacológico , Precursores de Ácido Nucleico/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Replicación del ADN , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteína 1 que Contiene Dominios SAM y HD/genéticaRESUMEN
Even the simplest organisms are too complex to have spontaneously arisen fully formed, yet precursors to first life must have emerged ab initio from their environment. A watershed event was the appearance of the first entity capable of evolution: the Initial Darwinian Ancestor. Here, we suggest that nucleopeptide reciprocal replicators could have carried out this important role and contend that this is the simplest way to explain extant replication systems in a mathematically consistent way. We propose short nucleic acid templates on which amino-acylated adapters assembled. Spatial localization drives peptide ligation from activated precursors to generate phosphodiester-bond-catalytic peptides. Comprising autocatalytic protein and nucleic acid sequences, this dynamical system links and unifies several previous hypotheses and provides a plausible model for the emergence of DNA and the operational code.
Asunto(s)
Modelos Químicos , Precursores de Ácido Nucleico/metabolismo , Nucleótidos/metabolismo , Origen de la Vida , Péptidos/metabolismo , PolimerizacionRESUMEN
Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions.
Asunto(s)
Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , ADN/metabolismo , Precursores de Ácido Nucleico/metabolismo , Nucleótidos/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , Pirofosfatasas/metabolismo , ADN/química , ADN/genética , Daño del ADN , Enzimas Reparadoras del ADN/genética , Genoma Humano , Inestabilidad Genómica , Humanos , Precursores de Ácido Nucleico/química , Precursores de Ácido Nucleico/genética , Nucleótidos/química , Oxidación-Reducción , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/genética , Pirofosfatasas/genética , Hidrolasas NudixRESUMEN
The SMN complex assembles Sm cores on snRNAs, a key step in the biogenesis of snRNPs, the spliceosome's major components. Here, using SMN complex inhibitors identified by high-throughput screening and a ribo-proteomic strategy on formaldehyde crosslinked RNPs, we dissected this pathway in cells. We show that protein synthesis inhibition impairs the SMN complex, revealing discrete SMN and Gemin subunits and accumulating an snRNA precursor (pre-snRNA)-Gemin5 intermediate. By high-throughput sequencing of this transient intermediate's RNAs, we discovered the previously undetectable precursors of all the snRNAs and identified their Gemin5-binding sites. We demonstrate that pre-snRNA 3' sequences function to enhance snRNP biogenesis. The SMN complex is also inhibited by oxidation, and we show that it stalls an inventory-complete SMN complex containing pre-snRNAs. We propose a stepwise pathway of SMN complex formation and snRNP biogenesis, highlighting Gemin5's function in delivering pre-snRNAs as substrates for Sm core assembly and processing.
Asunto(s)
Precursores de Ácido Nucleico/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas del Complejo SMN/metabolismo , Sitios de Unión , Células Cultivadas , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas Nucleares Pequeñas/metabolismoRESUMEN
The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.
Asunto(s)
Células Eucariotas/metabolismo , Precursores de Ácido Nucleico/metabolismo , Pirimidinas/metabolismo , Saccharomyces/clasificación , Saccharomyces/enzimología , Amidohidrolasas/química , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Saccharomyces/genética , Saccharomyces/metabolismoRESUMEN
Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.
Asunto(s)
ADN Mitocondrial/metabolismo , Enzimas/deficiencia , Enzimas/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Animales , ADN Mitocondrial/genética , Enzimas/metabolismo , Humanos , Enfermedades Mitocondriales/enzimología , Precursores de Ácido Nucleico/metabolismoAsunto(s)
Daño del ADN/genética , Enzimas Reparadoras del ADN , Mutación/genética , Precursores de Ácido Nucleico , 8-Hidroxi-2'-Desoxicoguanosina , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Enzimas Reparadoras del ADN/fisiología , ADN Polimerasa Dirigida por ADN/fisiología , Nucleótidos de Desoxiguanina/genética , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/fisiología , Proteínas de Escherichia coli/fisiología , GTP Ciclohidrolasa/fisiología , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Pirofosfatasas/fisiologíaRESUMEN
Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1-6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date.
Asunto(s)
Células Eucariotas/metabolismo , Precursores de Ácido Nucleico/metabolismo , Pirimidinas/metabolismo , Saccharomyces , Uracilo/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/química , Ácido Láctico/metabolismo , Estructura Molecular , Mutagénesis Sitio-Dirigida , Oxígeno/metabolismo , Pentosiltransferasa/metabolismo , Pirimidinas/química , Saccharomyces/genética , Saccharomyces/metabolismo , Uracilo/química , Urea/metabolismo , Uridina/metabolismoRESUMEN
How well do we understand which enzymes are involved in the primary metabolism of the cell? A recent study using comparative genomics and postgenomics approaches revealed a novel pathway in the most studied organism, Escherichia coli. The analysis of a new operon consisting of seven previously uncharacterized genes thought to be involved in the degradation of nucleic acid precursors shows the impact of comparative genomics on the discovery of novel pathways and enzymes.
Asunto(s)
Escherichia coli/enzimología , Genómica , Escherichia coli/genética , Genoma Bacteriano , Redes y Vías Metabólicas , Modelos Biológicos , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Operón , Uracilo/metabolismoRESUMEN
Among the human diseases that result from abnormalities in mitochondrial genome stability or maintenance are several that result from mutations affecting enzymes of deoxyribonucleoside triphosphate (dNTP) metabolism. In addition, it is evident that the toxicity of antiviral nucleoside analogs is determined in part by the extent to which their intracellular conversion to dNTP analogs occurs within the mitochondrion. Finally, recent work from this laboratory has shown considerable variation among different mammalian tissues with respect to mitochondrial dNTP pool sizes and has suggested that natural asymmetries in mitochondrial dNTP concentrations may contribute to the high rates at which the mitochondrial genome undergoes mutation. These factors suggest that much more information is needed about maintenance and regulation of dNTP pools within mammalian mitochondria. This review summarizes our current understanding and suggests directions for future research.
Asunto(s)
Replicación del ADN , ADN Mitocondrial/genética , Encefalomiopatías Mitocondriales/genética , Precursores de Ácido Nucleico/metabolismo , Encefalopatías/genética , Citosol/metabolismo , Desoxirribonucleótidos/metabolismo , Humanos , Mitocondrias , Modelos Biológicos , Oftalmoplejía/genéticaAsunto(s)
Arabidopsis/metabolismo , MicroARNs/metabolismo , ARN de Planta/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , MicroARNs/genética , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Plantas Modificadas Genéticamente , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN de Planta/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismoRESUMEN
Mammals have four deoxyribonucleoside kinases, the cytoplasmic (TK1) and mitochondrial (TK2) thymidine kinases, and the deoxycytidine (dCK) and deoxyguanosine (dGK) kinases, which salvage the precursors for nucleic acids synthesis. In addition to the native deoxyribonucleoside substrates, the kinases can phosphorylate and thereby activate a variety of anti-cancer and antiviral prodrugs. Recently, the crystal structure of human TK1 has been solved and has revealed that enzymes with fundamentally different origins and folds catalyze similar, crucial cellular reactions.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Humanos , Modelos Moleculares , Familia de Multigenes , Precursores de Ácido Nucleico/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/clasificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The mutation rate of the mammalian mitochondrial genome is higher than that of the nuclear genome. Because mitochondrial and nuclear deoxyribonucleoside triphosphate (dNTP) pools are physically distinct and because dNTP concentrations influence replication fidelity, we asked whether mitochondrial dNTP pools are asymmetric with respect to each other. We report here that the concentrations of the four dNTPs are not equal in mitochondria isolated from several tissues of both young and old rats. In particular, in most tissues examined, mitochondrial dGTP concentrations are high relative to the other dNTPs. Moreover, in the presence of the biased dNTP concentrations measured in heart and skeletal muscle, the fidelity of DNA synthesis in vitro by normally highly accurate mtDNA polymerase gamma is reduced, with error frequencies increased by as much as 3-fold, due to increased formation of template T.dGTP mismatches that are inefficiently corrected by proofreading. These data, plus some published data on specific mitochondrial mutations seen in human diseases, are consistent with the hypothesis that normal intramitochondrial dNTP pool asymmetries may contribute to spontaneous mutagenesis in the mammalian mitochondrial genome.
Asunto(s)
ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mutagénesis , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Animales , Encéfalo/metabolismo , ADN Polimerasa gamma , Replicación del ADN , ADN Mitocondrial/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleótidos/metabolismo , Humanos , Técnicas In Vitro , Masculino , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/metabolismo , Mitocondrias Musculares/metabolismo , Modelos Genéticos , Precursores de Ácido Nucleico/química , Ratas , Ratas Endogámicas F344 , Ratas WistarRESUMEN
Deoxyribonucleotides, monomers of macromolecular DNA and the chemical matter of genes, have received surprisingly little attention among chemists and molecular biologists alike, although their origin, properties, and mechanism of enzyme-catalyzed formation bear unique chemical traits which are the basis of DNA replication. Apart from providing insights in bioorganic free radical chemistry, present interest in deoxyribonucleotides stems from the expected demand of hundreds of kilograms per year for DNA chips and antisense constructs used in gene therapy, difficult to produce by conventional methods. A novel approach towards deoxyribonucleotide, and hence DNA formation in a putative primordial 'RNA world' has also recently emerged.
Asunto(s)
ADN/química , Desoxirribonucleótidos/química , Precursores de Ácido Nucleico/química , Animales , Biomimética , Catálisis , ADN/aislamiento & purificación , Replicación del ADN , ADN sin Sentido/síntesis química , Desoxirribonucleótidos/biosíntesis , Desoxirribosa/química , Evolución Molecular , Terapia Genética , Precursores de Ácido Nucleico/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/clasificación , Ribonucleótido Reductasas/metabolismoRESUMEN
BACKGROUND: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity. RESULTS: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA. It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end. When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3). We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes. Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific. CONCLUSIONS: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site.
Asunto(s)
Endorribonucleasas/fisiología , Escherichia coli/enzimología , Escherichia coli/genética , Ribosomas/fisiología , Composición de Base/genética , Secuencia de Bases/genética , Sitios de Unión/genética , Sitios de Unión/fisiología , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Precursores de Ácido Nucleico/fisiología , Procesamiento Postranscripcional del ARN/genética , Procesamiento Postranscripcional del ARN/fisiología , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Bacteriano/fisiología , ARN Ribosómico 23S/genética , ARN Ribosómico 23S/metabolismo , ARN Ribosómico 23S/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Ribonucleasa III , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
Pre-mRNA splicing has to be coordinated with other processes occurring in the nucleus including transcription, mRNA 3' end formation and mRNA export. To analyze the relationship between transcription and splicing, we constructed a network of nested introns. Introns were inserted in the 5' splice site and/or branchpoint of a synthetic yeast intron interrupting a reporter gene. The inserted introns mask the recipient intron from the cellular machinery until they are removed by splicing. Production of functional mRNA from these constructs therefore requires recognition of a spliced RNA as a splicing substrate. We show that recurrent splicing occurs in a sequential and ordered fashion in vivo. Thus, in Saccharomyces cerevisiae, intron recognition and pre-spliceosome assembly is not tightly coupled to transcription.
Asunto(s)
Genes Fúngicos , Intrones/genética , Precursores de Ácido Nucleico/metabolismo , Empalme del ARN , ARN de Hongos/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Conformación de Ácido Nucleico , Precursores de Ácido Nucleico/genética , Sondas de Oligonucleótidos , Plásmidos , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Bas?e-pairing between the terminal loops of helices P2.1 and P9.1a (P13) and P2 and P5c (P14) stabilize the folded structure of the Tetrahymena group I intron. Using native gel electrophoresis to analyze the folding kinetics of a natural pre-RNA containing the Tetrahymena intron, we show that P13 and P14 are the only native loop-loop interactions among six possible combinations. Other base-pairing interactions of the loop sequences stabilize misfolded and inactive pre-RNAs. Mismatches in P13 or P14 raised the midpoints and decreased the cooperativity of the Mg(2+)-dependent eqXuilibrium folding transitions. Although some mutations in P13 resulted in slightly higher folding rates, others led to slower folding compared to the wild-type, suggesting that P13 promotes formation of P3 and P7. In contrast, mismatches in P14 increased the rate of folding, suggesting that base-pairing between P5c and P2 stabilizes intermediates in which the catalytic core is misfolded. Although the peripheral helices stabilize the native structure of the catalytic core, our results show that formation of long-range interactions, and competition between correct and incorrect loop-loop base-pairs, decrease the rate at which the active pre-RNA structure is assembled.
Asunto(s)
Conformación de Ácido Nucleico , ARN Protozoario/química , Tetrahymena/química , Animales , Secuencia de Bases , Unión Competitiva , Intrones , Cinética , Magnesio/metabolismo , Modelos Moleculares , Mutación , Precursores de Ácido Nucleico/química , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Empalme del ARN , Estabilidad del ARN , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Protozoario/genética , ARN Protozoario/metabolismo , Tetrahymena/genética , Tetrahymena/metabolismoRESUMEN
The variety of biogenesis pathways for small nucleolar RNAs (snoRNAs) reflects the diversity of their genomic organization. We have searched for yeast snoRNAs which are affected by the depletion of the yeast ortholog of bacterial RNase III, Rnt1. In a yeast strain inactivated for RNT1, almost half of the snoRNAs tested are depleted with significant accumulation of monocistronic or polycistronic precursors. snoRNAs from both major families of snoRNAs (C/D and H/ACA) are affected by RNT1 disruption. In vitro, recombinant Rnt1 specifically cleaves pre-snoRNA precursors in the absence of other factors, generating intermediates which require the action of other enzymes for processing to the mature snoRNA. Most Rnt1 cleavage sites fall within potentially double-stranded regions closed by tetraloops with a novel consensus sequence AGNN. These results demonstrate that biogenesis of a large number of snoRNAs from the two major families of snoRNAs requires a common RNA endonuclease and a putative conserved structural motif.
Asunto(s)
Endorribonucleasas/metabolismo , ARN de Hongos/metabolismo , ARN Nuclear Pequeño/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Nucléolo Celular/metabolismo , Secuencia de Consenso , Endorribonucleasas/genética , Genes Fúngicos , Datos de Secuencia Molecular , Peso Molecular , Conformación de Ácido Nucleico , Precursores de Ácido Nucleico/química , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Sondas de Oligonucleótidos/genética , Caperuzas de ARN , Procesamiento Postranscripcional del ARN , ARN de Hongos/química , ARN de Hongos/genética , ARN Nuclear Pequeño/química , ARN Nuclear Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa III , Saccharomyces cerevisiae/genéticaRESUMEN
Taq and Tth DNA polymerases catalyzed polymerization of dATP and dTTP into poly d(A-T) without requiring added primer/template (Hanaki et al., Biochem. Biophys. Res. Commun. 238, 113-118), while the Stoffel fragment of Taq DNA polymerase and delta Tth DNA polymerase with respective deletions of ca. 290 and 250 N-terminal amino acids did not. The primer/template-independent polymerization appeared to proceed via two reactions, the slow process of formation of 16-19 nt long oligo d(A-T) without primer/template and the rapid process of elongation of the oligo d(A-T) by self-priming. As the former step was more sensitive to N-ethylmaleimide than the elongation reaction, probably the formation of the oligonucleotide preceded the elongation. But when the substrates were depleted, Taq DNA polymerase degraded the high molecular weigh d(A-T) polymer to the oligomers which were resistant to the further digestion by the 5'-->3' exonuclease activity of Taq DNA polymerase. Probably, the elongation and the degradation reactions proceeded simultaneously, the former process being faster than the latter in the presence of enough dATP and dTTP.
Asunto(s)
Cartilla de ADN/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Polímeros/metabolismo , Polimerasa Taq/metabolismo , Nucleótidos de Timina/metabolismo , ADN/antagonistas & inhibidores , ADN/biosíntesis , ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Etilmaleimida/farmacología , Peso Molecular , Precursores de Ácido Nucleico/antagonistas & inhibidores , Precursores de Ácido Nucleico/biosíntesis , Precursores de Ácido Nucleico/metabolismo , Oligonucleótidos/antagonistas & inhibidores , Oligonucleótidos/biosíntesis , Oligonucleótidos/metabolismo , Temperatura , Moldes GenéticosRESUMEN
The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.