RESUMEN

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.

Asunto(s)

Proteínas Arqueales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN Polimerasa beta/química , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , ADN/metabolismo , ADN Polimerasa beta/metabolismo , Cartilla de ADN/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/metabolismo , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteína de Replicación C , Subtilisina/metabolismo , Sulfolobus , Resonancia por Plasmón de Superficie , Factores de Tiempo

Asunto(s)

Methanococcus/enzimología , Nicotinamida-Nucleótido Adenililtransferasa/química , Nicotinamida-Nucleótido Adenililtransferasa/genética , Secuencia de Aminoácidos , Cromatografía/métodos , Durapatita , Estabilidad de Enzimas , Calor , Cinética , Methanococcus/genética , Methanococcus/crecimiento & desarrollo , Datos de Secuencia Molecular , Peso Molecular , Nicotinamida-Nucleótido Adenililtransferasa/aislamiento & purificación , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Sistemas de Lectura Abierta , Subunidades de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sulfolobus/enzimología , Sulfolobus/genética , Termodinámica

RESUMEN

The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387-->Phe, Tyr387-->Trp, Tyr387-->His, Tyr387-->Asn and Tyr387-->Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis.

Asunto(s)

ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Mutación , Reacción en Cadena de la Polimerasa/métodos , Ingeniería de Proteínas , Thermococcus/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Fagos de Bacillus/enzimología , Secuencia Conservada/genética , Cristalografía por Rayos X , ADN/biosíntesis , ADN/genética , ADN Polimerasa beta/genética , ADN Polimerasa beta/aislamiento & purificación , Exonucleasas/química , Exonucleasas/genética , Exonucleasas/metabolismo , Cinética , Operón Lac/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sulfolobus/enzimología

RESUMEN

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed.

Asunto(s)

Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Sulfolobus , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Replicación del ADN , ADN de Archaea/genética , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Genes Arqueales/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/aislamiento & purificación , Holoenzimas/metabolismo , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Peso Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteína de Replicación C , Alineación de Secuencia , Homología de Secuencia , Sulfolobus/química , Sulfolobus/enzimología , Sulfolobus/genética

RESUMEN

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.

Asunto(s)

ADN Primasa/aislamiento & purificación , Methanococcus/enzimología , Secuencia de Aminoácidos , ADN Primasa/genética , ADN Primasa/metabolismo , Estabilidad de Enzimas , Escherichia coli , Concentración de Iones de Hidrógeno , Methanococcus/genética , Methanococcus/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

RESUMEN

Herein, we report the identification and characterization of two DNA polymerase processivity factors from the thermoacidophilic archaeon Sulfolobus solfataricus. They, referred to as 039p (244 amino acid residues, 27 kDa) and 048p (249 amino acid residues, 27 kDa), present significant primary structure similarity to eukaryotic proliferating cell nuclear antigen (PCNA). We demonstrate that both 039p and 048p form oligomers in solution and are able to substantially activate the synthetic activity of the single-subunit family B DNA polymerase from S. solfataricus (Sso DNA pol B1) on poly(dA)-oligo(dT) as a primer-template. This stimulatory effect is the result of enhanced DNA polymerase processivity, as indicated by the analysis of the elongation products on polyacrylamide gels. Activation of Sso DNA pol B1 synthetic activity was also observed on linear primed single-stranded M13 mp18 DNA as a template. By immunoblot analysis using specific rabbit antisera, 039p and 048p were both detected in the logarithmic and stationary phases of S. solfataricus growth curve. This is the first report of the identification and biochemical characterization of two distinct DNA polymerase processivity factors from the same organism. The significance of these findings for the understanding of the DNA replication process in Archaea is discussed.

Asunto(s)

Proteínas Arqueales/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Sulfolobus/enzimología , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Clonación Molecular , Replicación del ADN , Escherichia coli , Humanos , Datos de Secuencia Molecular , Antígeno Nuclear de Célula en Proliferación/química , Conejos , Homología de Secuencia de Aminoácido , Sulfolobus/genética , Sulfolobus/metabolismo

RESUMEN

Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating. Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required. In this context the study of NMN adenylyltransferase (EC 2.7.7.1),. a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance. We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus. The determination of partial sequence of the S. solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii, allowed us, based on sequence similarity, to identify the M. jannaschii NMN adenylyltransferase gene. As far as we know from literature, this is the first report on the NMN adenylyltransferase gene.

Asunto(s)

Nicotinamida-Nucleótido Adenililtransferasa/genética , Sulfolobus/enzimología , Sulfolobus/genética , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

The thermophilic and thermostable family B DNA polymerase from the archaeon Sulfolobus solfataricus (Mr of about 100 kDa) has been crystallized by the hanging-drop vapour-diffusion method at 294 K using ammonium sulfate as precipitant. The crystals belong to the monoclinic space group C2 with cell dimensions a = 187.4, b = 68.5, c = 125.8 A and beta = 107.8 degrees and diffract up to 2.7 A resolution on a rotating-anode X-ray source. Native data have been collected at 100 K. A heavy-atom derivative search is in progress.

Asunto(s)

Proteínas Bacterianas/química , ADN Polimerasa Dirigida por ADN/química , Conformación Proteica , Sulfolobus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN/aislamiento & purificación

RESUMEN

Herein, we report on the mutational analysis of a 70-amino acid segment (region 1, residues 438-508) of family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol). Region 1, which lies between the Exo III sequence and the similarity motif D- -SLYP, connects the exonuclease and polymerase domains of Sso DNA pol. Two C-terminally deleted forms of the enzyme, proteins N438 (residues 1-438) and N508 (residues 1-508), were overproduced in the recombinant form and biochemically characterized. They contain the three evolutionarily conserved Exo motifs, but differ in the extent of the C-terminal deletion, since only N508 includes region 1. Both have been found to retain a Mn2+-dependent 3'-5' exonuclease activity, whose thermal stability appears to be increased in comparison to that of the full-sized enzyme. Assays for processive 3'-5' exonuclease activity, carried out with the heparin trap method on a 24-base oligonucleotide, have revealed that protein N508, as well as the full-length Sso DNA pol, retains a level of processivity of the degradative function substantially higher than that for protein N438. In addition, six site-specific mutations have been introduced at the highly conserved Y-GG/A motif, which has been found within Sso DNA pol region 1. All mutant proteins (Lys491Ile, Tyr495Ser, Lys496Ile, Gly497Ala, and Ala498Val) display increased processivity of their 3'-5' exonuclease activity, with the exception of protein Tyr495Phe. By a steady-state kinetic analysis of the exonucleolytic reaction on a 24-base oligonucleotide, the above site-specific mutations have been found to affect Km values consistently with the observed differences in the processivity values, whereas the effect on the kcat values seems to be less important. The results from this mutational analysis indicate that region 1 is involved in determining the processivity of the proofreading function, directly interacting with the nucleic acid substrate.

Asunto(s)

Aminoácidos/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Procesamiento Proteico-Postraduccional , Sulfolobus/enzimología , Bacteriófago T4/enzimología , ADN Bacteriano/genética , ADN Polimerasa Dirigida por ADN/genética , Activación Enzimática/genética , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/genética , Cinética , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Sulfolobus/genética

RESUMEN

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3'-5' exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction.

Asunto(s)

ADN Polimerasa Dirigida por ADN/química , Sulfolobus/enzimología , Dominio Catalítico , Mapeo Cromosómico , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Polimerasa Dirigida por ADN/clasificación , ADN Polimerasa Dirigida por ADN/genética , Genes Arqueales , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sulfolobus/genética

RESUMEN

The sequences of a number of archaeal genomes have recently been completed, and many more are expected shortly. Consequently, the research of Archaea in general and hyperthermophiles in particular has entered a new phase, with many exciting discoveries to be expected. The wealth of sequence information has already led, and will continue to lead to the identification of many enzymes with unique properties, some of which have potential for industrial applications. Subsequent functional genomics will help reveal fundamental matters such as details concerning the genetic, biochemical and physiological adaptation of extremophiles, and hence give insight into their genomic evolution, polypeptide structure-function relations, and metabolic regulation. In order to optimally exploit many unique features that are now emerging, the development of genetic systems for hyperthermophilic Archaea is an absolute requirement. Such systems would allow the application of this class of Archaea as so-called "cell factories": (i) expression of certain archaeal enzymes for which no suitable conventional (mesophilic bacterial or eukaryal) systems are available, (ii) selection for thermostable variants of potentially interesting enzymes from mesophilic origin, and (iii) the development of in vivo production systems by metabolic engineering. An overview is given of recent insight in the molecular biology of hyperthermophilic Archaea, as well as of a number of promising developments that should result in the generation of suitable genetic systems in the near future.

Asunto(s)

Archaea/genética , Regulación Bacteriana de la Expresión Génica/genética , Thermus/genética , Archaea/química , Archaea/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Replicación del ADN , Genotipo , Datos de Secuencia Molecular , Familia de Multigenes , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Thermus/química , Thermus/metabolismo , Factores de Transcripción/química , Transcripción Genética

RESUMEN

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.

Asunto(s)

Bacillus/enzimología , Esterasas/química , Esterol Esterasa/química , Aminoácidos/análisis , Proteínas Bacterianas/química , Sitios de Unión/fisiología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/genética , Escherichia coli/genética , Expresión Génica , Concentración de Iones de Hidrógeno , Cinética , Conformación Molecular , Estructura Molecular , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura

RESUMEN

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the synthesis of NAD+ and nicotinic acid adenine dinucleotide. It has been purified to homogeneity from cellular extracts of the thermophilic archaeon Sulfolobus solfataricus. Through a database search, a highly significant match was found between its N-terminal sequence and a hypothetical protein coded by the thermophilic archaeon Methanococcus jannaschii MJ0541 open reading frame (GenBank accession no. U67503). The MJ0541 gene was isolated, cloned into a T7-based vector, and expressed in Escherichia coli cells, yielding a high level of thermophilic NMN adenylyltransferase activity. The expressed protein was purified to homogeneity by a single-step chromatographic procedure. Both the subunit molecular mass and the N-terminal sequence of the pure recombinant protein were as expected from the deduced amino acid sequence of the MJ0541 open reading frame-encoded protein. Molecular and kinetic properties of the enzymes from both archaea are reported and compared with those already known for the mesophilic eukaryotic NMN adenylyltransferase.

Asunto(s)

Proteínas Arqueales/aislamiento & purificación , Methanococcus/enzimología , Nicotinamida-Nucleótido Adenililtransferasa/aislamiento & purificación , Sulfolobus/enzimología , Secuencia de Aminoácidos , Aminoácidos/análisis , Proteínas Arqueales/genética , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Calor , Methanococcus/genética , Datos de Secuencia Molecular , Nicotinamida-Nucleótido Adenililtransferasa/genética , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol) is a monomer of about 100 kDa with two associated catalytic functions: 3'-5' exonuclease and DNA polymerase activities. The structure of this enzyme in the free and DNA-bound states was probed by limited proteolysis and fluorescence spectroscopy measurements. The results of partial trypsin proteolysis experiments on the recombinant Sso DNA pol pinpointed three major sites of protease sensitivity: near the N-terminus, within the center, and near the C-terminal end of the polypeptide chain. When partial trypsin digestion was carried out in the presence of either activated calf thymus DNA or primed M13mp18 single-stranded DNA, changes in cleavage pattern and in susceptibility to protease were detected. This phenomenon was dependent on the nucleic acid concentration and suggested the occurrence of DNA-induced conformational changes. These were also probed by steady-state fluorescence spectroscopy measurements using acrylamide as a quencher. Fine mapping of the DNA-specific cleavage sites allowed us to precisely locate the protein subdomains which were affected by these structural changes. Importantly, a specific proteolytic fragment of about 8 kDa was recovered after partial digestion of Sso DNA pol only in the presence of nucleic acid ligands. It was found to start at residues 392-394 and to span the protease-hypersensitive central region of the polypeptide chain. Its involvement in critical polymerase functions, such as substrate binding and/or enzyme processivity, was discussed. In addition, we found that controlled trypsin digestion of Sso DNA pol did not inactivate either polymerase or 3'-5' exonuclease activity concomitantly with the disappearance of full-sized enzyme. Activity gel analysis revealed that proteolytic products corresponding to the amino- and carboxyl-terminal halves of the enzyme retained 3'-5' exonuclease and DNA polymerase activity, respectively. These results are in line with the model of modular organization proposed for Sso DNA pol in a previous report.

Asunto(s)

ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Conformación Proteica , Sulfolobus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ADN de Cadena Simple/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Tripsina/metabolismo , Triptófano/metabolismo

RESUMEN

The molecular biology of extremophiles has recently attracted much interest, both in terms of cell adaptation to extreme environmental conditions and the development of manipulative genetic techniques. Although molecular genetic techniques have been successfully applied to halophiles and methanogens, their use with hyperthermophiles is limited by the extreme growth conditions that these organisms require. Much information on the thermophilic Archaea, has been obtained by studying the key enzymes involved in fundamental cell processes, such as transcription and replication, and by the cloning, sequence comparison and heterologous expression of structural genes. The development of viral vectors and systems for transformation, mutant production and screening will permit increased genetic manipulation of these organisms.

RESUMEN

In this study we report on the evidence that an alpha-like DNA polymerase purified from the thermoacidophilic archaeon Sulfolobus solfataricus has a modular organization of its associated catalytic activities (polymerase and 3'-5' exonuclease). This enzyme, a monomer of about 100 kDa whose complete primary structure is available, has a protease hypersensitive site that is likely to be cleaved by the action of endogenous proteases during the purification procedure. As a consequence of that, two proteolytic fragments of about 50 and 40 kDa, in addition to the intact 100-kDa molecular species, can be detected upon SDS-PAGE of highly purified S. solfataricus DNA polymerase samples. The amino-terminal microsequence analysis by Edman degradation has revealed that the 50- and the 40-kDa polypeptides correspond to the carboxyl- and the amino-terminal portion of the protein molecule, respectively. Using the bidimensional activity gel assay procedure, recently described by Longley and Mosbaugh (Longley, M. J., and Mosbaugh, D. W. (1991) Biochemistry 30, 2655-2664), we have demonstrated that the 50-kDa fragment retains a Mg(2+)-dependent DNA polymerizing activity, whereas the 40-kDa polypeptide is able to catalyze the excision of mispaired nucleotides at the 3'-OH terminus of a primer/template DNA substrate in the presence of Mn2+ ions. On the other hand, the 100-kDa protein possess both activities. To date, this is the first report indicating, on the basis of direct functional data, that the polymerization and the 3'-5' exonuclease activity of a family B DNA polymerase can be ascribed to physically distinct modules of the enzyme molecule.

Asunto(s)

ADN Polimerasa II/química , ADN Polimerasa II/metabolismo , Exodesoxirribonucleasas/metabolismo , Sulfolobus/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , ADN Polimerasa II/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/aislamiento & purificación , Fibrinógeno/metabolismo , Sustancias Macromoleculares , Magnesio/farmacología , Manganeso/farmacología , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Especificidad por Sustrato

RESUMEN

The gene encoding the thermostable DNA polymerase from the archaeon Sulfolobus solfataricus (strain MT 4) was isolated by means of two degenerate oligonucleotide probes. They were designed on the basis of partial enzyme amino acid sequences. The gene was found to encode a 882 residues polypeptide chain with a deduced molecular mass of about 100 kDa. By comparison with other archaeal genes, putative regulatory sites were identified in the gene-flanking regions. By computer-assisted homology search, several sequence similarities among S. solfataricus and family B DNA polymerases were found. In addition, conserved sequence motifs, implicated in the 3'-5' exonuclease activity of E. coli DNA polymerase I and shared by various family A and B DNA polymerases, were also identified. This result suggests that the proofreading domains of all these enzymes are evolutionarily related.

Asunto(s)

ADN Polimerasa Dirigida por ADN/genética , Genes Bacterianos , Sulfolobus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Sulfolobus/enzimología

RESUMEN

A DNA-dependent DNA polymerase was obtained in homogenous form from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme, purified 706-fold, has a molecular mass of about 110000 daltons as determined by gel filtration and by glycerol gradient centrifugation. It requires Mg++ for its activity and has a pH optimum of 7.7. The activity is sharply dependent on the ionic strength. The enzyme is thermostable; its properties and activity requirements were characterized. The features of this enzyme are compared to those of other DNA polymerases isolated either from prokaryotes or eukaryotes.

Asunto(s)

Archaea/enzimología , Bacterias/enzimología , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/metabolismo , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Manganeso/farmacología , Peso Molecular , Inhibidores de la Síntesis del Ácido Nucleico , Especificidad por Sustrato , Temperatura

RESUMEN

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.

Asunto(s)

Archaea/enzimología , Bacterias/enzimología , Galactosidasas/aislamiento & purificación , Calor , beta-Galactosidasa/aislamiento & purificación , Aminoácidos/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Reactivos de Enlaces Cruzados , Electroforesis/métodos , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Inmunoquímica , Focalización Isoeléctrica , Cinética

RESUMEN

An NAD+-dependent alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was detected in cellular extracts of the extreme thermophilic archaebacterium Sulfolobus solfataricus. The enzyme was purified to homogeneity and shown to be a dimer with a native molecular mass of 71 kDa by sucrose gradient centrifugation and SDS electrophoresis. The enzyme has a broad substrate specificity that includes linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones and anisaldehyde. The enzyme has an extraordinary thermophilicity and a remarkable thermostability, and appears to have some properties and a structure different from those previously described for thermophilic alcohol dehydrogenases.

Asunto(s)

Alcohol Deshidrogenasa/aislamiento & purificación , Archaea/enzimología , Bacterias/enzimología , Alcohol Deshidrogenasa/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Especificidad por Sustrato