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A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen ArchiMer
Mesiet Cladiere, L; Norais, Cédric; Kuhn, Joelle; Briffotaux, Julien; Sloostra, Jerry W.; Ferrari, Elena; Hubscher, Ulrich; Flament, Didier; Myllykallio, Hannu.
During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human...
Tipo: Text Palavras-chave: Ribonuclease HII; Combinatorial peptide synthesis; PCNA.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-3297.pdf
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Alpha-agarases define a new family of glycoside hydrolases, distinct from beta-agarase families ArchiMer
Flament, Didier; Barbeyron, Tristan; Jam, Murielle; Potin, Philippe; Czjzek, Mirjam; Kloareg, Bernard; Michel, Gurvan.
The gene encoding the alpha-agarase from "Alteromonas agarilytica" (proposed name) has been cloned and sequenced. The gene product (154 kDa) is unrelated to Beta-agarases and instead belongs to a new family of glycoside hydrolases (GH96). The -agarase also displays a complex modularity, with the presence of five thrombospondin type 3 repeats and three carbohydrate-binding modules.
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Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-2826.pdf
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An Extended Network of Genomic Maintenance in the Archaeon Pyrococcus abyssi Highlights Unexpected Associations between Eucaryotic Homologs ArchiMer
Pluchon, Pierre Francois; Fouqueau, Thomas; Creze, Christophe; Laurent, Sebastien; Briffotaux, Julien; Hogrel, Gaelle; Palud, Adeline; Henneke, Ghislaine; Godfroy, Anne; Hausner, Winfried; Thomm, Michael; Nicolas, Jacques; Flament, Didier.
In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA...
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Ano: 2013 URL: http://archimer.ifremer.fr/doc/00171/28198/26556.pdf
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Analysis of the first genome of a hyperthermophilic marine virus-like particle, PAV1, isolated from Pyrococcus abyssi ArchiMer
Geslin, Claire; Gaillard, Melusine; Flament, Didier; Rouault, Karen; Le Romancer, Marc; Prieur, Daniel; Erauso, Gael.
Only one virus-like particle (VLP) has been reported from hyperthermophilic Euryarchaeotes. This VLP, named PAV1 is shaped like a lemon and was isolated from a strain of "Pyrococcus abyssi," a deep-sea isolate. Its genome consists of a double-stranded circular DNA of 18 kb which is also present at a high copy number (60 per chromosome) free within the host cytoplasm but is not integrated into the host chromosome. Here, we report the results of complete analysis of the PAV1 genome. All the 25 predicted genes, except 3, are located on one DNA strand. A transcription map has been made by using a reverse transcription-PCR assay. All the identified open reading frames (ORFs) are transcribed. The most significant similarities relate to four ORFs. ORF 180a shows...
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Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-3536.pdf
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Binding to PCNA in Euryarchaeal DNA Replication Requires Two PIP Motifs for DNA Polymerase D and One PIP Motif for DNA Polymerase B ArchiMer
Castrec, Benoit; Rouillon, Christophe; Henneke, Ghislaine; Flament, Didier; Querellou, Joel; Raffin, Jean-paul.
Replicative DNA polymerases possess a canonical C-terminal proliferating cell nuclear antigen (PCNA)-binding motif termed the PCNA-interacting protein (PIP) box. We investigated the role of the PIP box on the functional interactions of the two DNA polymerases, PabPol B (family B) and PabPol D (family D), from the hyperthermophilic euryarchaeon Pyrococcus abyssi, with its cognate PCNA. The PIP box was essential for interactions of PabPol B with PCNA, as shown by surface plasmon resonance and primer extension studies. In contrast, binding of PabPol D to PCNA was affected only partially by removing the PIP motif. We identified a second palindromic PIP box motif at the N-terminus of the large subunit of PabPol D that was required for the interactions of PabPol...
Tipo: Text Palavras-chave: Archaea; PIP box; PCNA binding motifs; DNA polymerases; DNA replication.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-7317.pdf
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Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi ArchiMer
Zappa, Sebastien; Rolland, Jean-luc; Flament, Didier; Gueguen, Yannick; Boudrant, Joseph; Dietrich, Jacques.
This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degreesC, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degreesC of 18 and 5 h, respectively....
Tipo: Text Palavras-chave: Escherichia coli; Characterization; Isolation; Gene; Alkaline phosphatase; Pyrococcus abyssi; Archaeon.
Ano: 2001 URL: http://archimer.ifremer.fr/doc/2001/publication-1268.pdf
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Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi ArchiMer
Leulliot, N.; Quevillon-cheruel, S.; Graille, M.; Geslin, Claire; Flament, Didier; Le Romancer, Marc; Van Tilbeurgh, H..
Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 angstrom. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a...
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Ano: 2013 URL: http://archimer.ifremer.fr/doc/00134/24562/22595.pdf
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Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus ArchiMer
Birien, Tiphaine; Thiel, Axel; Henneke, Ghislaine; Flament, Didier; Moalic, Yann; Jebbar, Mohamed.
A gene disruption system for Thermococcus barophilus was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a pyrF gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in trans, but produces many false positives (<80%). To significantly reduce the rate of false positives, we used another counterselective marker, 6-methylpurine (6-MP), a toxic analog of adenine developed in Thermococcus kodakarensis, consistently correlated with the TK0664 gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced pyrF by TK0664 on our suicide vector and tested T. barophilus strain sensitivity to 6-MP...
Tipo: Text Palavras-chave: Archaea; Piezophiles; Hyperthermophiles; Genetics; Gene deletion; Deep sea; Hydrothermal vents.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00425/53679/54523.pdf
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DNA polymerase switching on homotrimeric PCNA at the replication fork of the euryarchaea Pyrococcus abyssi ArchiMer
Rouillon, Christophe; Henneke, Ghislaine; Flament, Didier; Querellou, Joel; Raffin, Jean-paul.
DNA replication in Archaea, as in other organisms, involves large protein complexes called replisomes. In the Euryarchaeota subdomain, only two putative replicases have been identified, and their roles in leading and lagging strand DNA synthesis are still poorly understood. In this study, we focused on the coupling of proliferating cell nuclear antigen (PCNA)loading mechanisms with DNA polymerase function in the Euryarchaea Pyrococcus abyssi. PCNA spontaneously loaded onto primed DNA, and replication factor C dramatically increased this loading. Surprisingly, the family B DNA polymerase (Pol B) also increased PCNA loading, probably by stabilizing the clamp on primed DNA via an essential motif. In contrast, on an RNA-primed DNA template, the PCNA/Pol B...
Tipo: Text Palavras-chave: RF C; PCNA loading; DNA polymerase switching; DNA replication; Archaea.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-2638.pdf
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Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus ArchiMer
Thiel, Axel; Michoud, Gregoire; Moalic, Yann; Flament, Didier; Jebbar, Mohamed.
In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single-and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (Delta pyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus...
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Ano: 2014 URL: http://archimer.ifremer.fr/doc/00187/29818/28263.pdf
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MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms ArchiMer
Groisillier, Agnes; Herve, Christiane; Jeudy, Alexandra; Rebuffet, Etienne; Pluchon, Pierre Francois; Chevolot, Yann; Flament, Didier; Geslin, Claire; Morgado, Isabel M.; Power, Deborah; Branno, Margherita; Moreau, Herve; Michel, Gurvan; Boyen, Catherine; Czjzek, Mirjam.
Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still...
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Ano: 2010 URL: http://archimer.ifremer.fr/doc/00011/12219/9003.pdf
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Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of elF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive ArchiMer
Laurent, Sebastien; Richard, Adrien; Mulner-lorillon, Odile; Morales, Julia; Flament, Didier; Glippa, Virginie; Bourdon, Jeremie; Gosselin, Pauline; Siegel, Anne; Cormier, Patrick; Belle, Robert.
Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koffi) of the elF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP Additionally, the changes in both processes should occur in 5 min time interval...
Tipo: Text Palavras-chave: Translational control; Sea urchin embryos; Mechanisms of fertilization; Deterministic model; Translation simulation.
Ano: 2014 URL: http://archimer.ifremer.fr/doc/00382/49341/49731.pdf
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Physical and functional interplay between PCNA DNA clamp and Mre11–Rad50 complex from the archaeon Pyrococcus furiosus ArchiMer
Hogrel, Gaelle; Lu, Yang; Laurent, Sebastien; Henry, Etienne; Etienne, Clarisse; Duy Khanh Phung,; Dulermo, Remi; Bosse, Audrey; Pluchon, Pierre Francois; Clouet-d'Orval, Beatrice; Flament, Didier.
Several archaeal species prevalent in extreme environments are particularly exposed to factors likely to cause DNA damages. These include hyperthermophilic archaea (HA), living at temperatures >70°C, which arguably have efficient strategies and robust genome guardians to repair DNA damage threatening their genome integrity. In contrast to Eukarya and other archaea, homologous recombination appears to be a vital pathway in HA, and the Mre11–Rad50 complex exerts a broad influence on the initiation of this DNA damage response process. In a previous study, we identified a physical association between the Proliferating Cell Nuclear Antigen (PCNA) and the Mre11–Rad50 (MR) complex. Here, by performing co-immunoprecipitation and SPR analyses, we identified a...
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Ano: 2018 URL: http://archimer.ifremer.fr/doc/00439/55092/56534.pdf
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Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain ArchiMer
Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joel; Dietrich, Jacques; Hubscher, Ulrich; Raffin, Jean-paul.
The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (Pab RFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the Pab RFC-small subunit could be purified, while the large subunit (Pab RFC-large) alone was completely insoluble. The highly purified Pab RFC complex possessed an...
Tipo: Text Palavras-chave: PCNA binding domain; Pyrococcus abyssi; Hyperthermophile; Archaea; Replication factor C.
Ano: 2002 URL: https://archimer.ifremer.fr/doc/2002/publication-675.pdf
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RNA processing machineries in Archaea: the 5′-3′ exoribonuclease aRNase J of the β-CASP family is engaged specifically with the helicase ASH-Ski2 and the 3′-5′ exoribonucleolytic RNA exosome machinery ArchiMer
Phung, Duy Khanh; Etienne, Clarisse; Batista, Manon; Langendijk-genevaux, Petra; Moalic, Yann; Laurent, Sebastien; Liuu, Sophie; Morales, Violette; Jebbar, Mohamed; Fichant, Gwennaele; Bouvier, Marie; Flament, Didier; Clouet-d’orval, Béatrice.
A network of RNA helicases, endoribonucleases and exoribonucleases regulates the quantity and quality of cellular RNAs. To date, mechanistic studies focussed on bacterial and eukaryal systems due to the challenge of identifying the main drivers of RNA decay and processing in Archaea. Here, our data support that aRNase J, a 5′-3′ exoribonuclease of the β-CASP family conserved in Euryarchaeota, engages specifically with a Ski2-like helicase and the RNA exosome to potentially exert control over RNA surveillance, at the vicinity of the ribosome. Proteomic landscapes and direct protein–protein interaction analyses, strengthened by comprehensive phylogenomic studies demonstrated that aRNase J interplay with ASH-Ski2 and a cap exosome subunit. Finally,...
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Ano: 2020 URL: https://archimer.ifremer.fr/doc/00610/72250/71056.pdf
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Structural insights into a new homodimeric self-activated GTPase family ArchiMer
Gras, S; Chaumont, V; Fernandez, B; Carpentier, P; Charrier Savournin, F; Schmitt, Sophie; Pineau, C; Flament, Didier; Hecker, A; Forterre, P; Armengaud, J; Housset, D.
The human XAB1/ MBDin GTPase and its close homologues form one of the ten phylogenetically distinct families of the SIMIBI ( after signal recognition particle, MinD and BioD) class of phosphate- binding loop NTPases. The genomic context and the partners identified for the archaeal and eukaryotic homologues indicate that they are involved in genome maintenance - DNA repair or replication. The crystal structure of PAB0955 from Pyrococcus abyssi shows that, unlike other SIMIBI class G proteins, these highly conserved GTPases are homodimeric, regardless of the presence of nucleotides. The nucleotide- binding site of PAB0955 is rather rigid and its conformation is closest to that of the activated SRP G domain. One insertion to the G domain bears a strictly...
Tipo: Text Palavras-chave: Structure; SIMIBI; Replication; GTPase; Crystal.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-4724.pdf
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Structure and function of a novel endonuclease acting on branched DNA substrates ArchiMer
Ren, Bin; Kuehn, Joelle; Meslet-cladiere, Laurence; Briffotaux, Julien; Norais, Cedric; Lavigne, Regis; Flament, Didier; Ladenstein, Rudolf; Myllykallio, Hannu.
We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two-domain dumbbell-like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non-catalytic ssDNA-binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double-stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3' and 5' extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute...
Tipo: Text Palavras-chave: Structure function studies; RecB family; Novel endonuclease; DNA repair; Branched DNA structures.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-6807.pdf
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The Glycine-Rich Motif of Pyrococcus abyssi DNA Polymerase D Is Critical for Protein Stability ArchiMer
Castrec, Benoit; Laurent, Sebastien; Henneke, Ghislaine; Flament, Didier; Raffin, Jean-paul.
A glycine-rich motif described as being involved in human polymerase 8 proliferating cell nuclear antigen (PCNA) binding has also been identified in all euryarchaeal DNA polymerase D (Pol D) family members. We redefined the motif as the (G)-PYF box. In the present study, Pol D (G)-PYF box motif mutants from Pyrococcus abyssi were generated to investigate its role in functional interactions with the cognate PCNA. We demonstrated that this motif is not essential for interactions between PabPol D (P. abyssi Pol D) and PCNA, using surface plasmon resonance and primer extension studies. Interestingly, the (G)-PYF box is located in a hydrophobic region close to the active site. The (G)-PYF box mutants exhibited altered DNA binding properties. In addition, the...
Tipo: Text Palavras-chave: DNA replication DNA polymerase D; (G)-PYF box Archaea thermostability.
Ano: 2010 URL: http://archimer.ifremer.fr/doc/00002/11293/7879.pdf
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The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: A multifunctional enzyme for initiation and repair? ArchiMer
Le Breton, Magali; Henneke, Ghislaine; Norais, C; Flament, Didier; Myllykallio, H; Querellou, Joel; Raffin, Jean-paul.
We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase alpha-primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and...
Tipo: Text Palavras-chave: Strand displacement; Gap filling; DNA primase; Archaea; DNA replication.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/publication-3520.pdf
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The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication ArchiMer
Henneke, Ghislaine; Flament, Didier; Hubscher, Ulrich; Querellou, Joel; Raffin, Jean-paul.
DNA polymerases carry out DNA synthesis during DNA replication, DNA recombination and DNA repair. During the past five years, the number of DNA polymerases in both eukarya and bacteria has increased to at least 19 and multiple biological roles have been assigned to many DNA polymerases. Archaea, the third domain of life, on the other hand, have only a subset of the eukaryotic-like DNA polymerases. The diversity among the archaeal DNA polymerases poses the intriguing question of their functional tasks. Here, we focus on the two identified DNA polymerases, the family B DNA polymerase B (PabpolB) and the family D DNA polymerase D (PabpolD) from the hyperthermophilic euryarchaeota Pyrococcus abyssi. Our data can be summarized as follows: (i) both Pabpols are...
Tipo: Text Palavras-chave: DNA polymerase; Strand displacement; Gap filling; Euryarchaea; DNA replication.
Ano: 2005 URL: http://archimer.ifremer.fr/doc/2005/publication-423.pdf
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