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| Registros recuperados: 24 | |
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| 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... |
| Tipo: Text |
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| Ano: 2013 |
URL: http://archimer.ifremer.fr/doc/00171/28198/26556.pdf |
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| 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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| Ralec, Celine; Henry, Etienne; Lemor, Melanie; Killelea, Tom; Henneke, Ghislaine. |
| Divalent metal ions, usually Mg2+, are required for both DNA synthesis and proofreading functions by DNA polymerases (DNA Pol). Although used as a non-reactive cofactor substitute for binding and crystallographic studies, Ca2+ supports DNA polymerization by only one DNA Pol, Dpo4. Here, we explore whether Ca2+-driven catalysis might apply to high-fidelity (HiFi) family B DNA Pols. The consequences of replacing Mg2+ by Ca2+ on base pairing at the polymerase active site as well as the editing of terminal nucleotides at the exonuclease active site of the archaeal Pyrococcus abyssi DNA Pol (PabPolB) are characterized and compared to other (families B, A, Y, X, D) DNA Pols. Based on primer extension assays, steady-state kinetics and ion-chased experiments, we... |
| Tipo: Text |
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| Ano: 2017 |
URL: http://archimer.ifremer.fr/doc/00405/51663/52210.pdf |
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| 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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| Lemor, Melanie; Kong, Ziqing; Henry, Etienne; Brizard, Raphael; Laurent, Sebastien; Bosse, Audrey; Henneke, Ghislaine. |
| Consistent with the fact that ribonucleotides (rNTPs) are in excess over deoxyribonucleotides (dNTPs) in vivo, recent findings indicate that replicative DNA polymerases (DNA Pols) are able to insert ribonucleotides (rNMPs) during DNA synthesis, raising crucial questions about the fidelity of DNA replication in both Bacteria and Eukarya. Here, we report that the level of rNTPs is 20-fold higher than that of dNTPs in Pyrococcus abyssi cells. Using dNTP and rNTP concentrations present in vivo, we recorded rNMP incorporation in a template-specific manner during in vitro synthesis, with the family-D DNA Pol (PolD) having the highest propensity compared with the family-B DNA Pol and the p41/p46 complex. We also showed that ribonucleotides accumulate at a... |
| Tipo: Text |
Palavras-chave: Archaea; DNA replication and repair; DNA polymerase; Nucleotide pool; Translesion synthesis. |
| Ano: 2018 |
URL: https://archimer.ifremer.fr/doc/00464/57603/59796.pdf |
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| 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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| Henneke, Ghislaine. |
| Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi. I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5'-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5'-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by... |
| Tipo: Text |
Palavras-chave: Archaea; DNA ligase; DNA polymerase; Nuclease; Okazaki fragment. |
| Ano: 2012 |
URL: http://archimer.ifremer.fr/doc/00107/21841/20923.pdf |
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| Palud, Adeline; Villani, Giuseppe; L'Haridon, Stephane; Querellou, Joel; Raffin, Jean-paul; Henneke, Ghislaine. |
| Spontaneous and induced abasic sites in hyperthermophiles DNA have long been suspected to occur at high frequency. Here, Pyrococcus abyssi was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. We demonstrated that endogenous AP sites persist at a slightly higher level in P. abyssi genome compared with Escherichia coli. Then, the two replicative DNA polymerases, PabpolB and PabpolD, were characterized in presence of DNA containing abasic sites. Both Pabpols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, PabpolB could incorporate in front of the damage and even replicate to the full-length oligonucleotides containing a specific AP site, but only when present at... |
| Tipo: Text |
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| Ano: 2008 |
URL: http://archimer.ifremer.fr/doc/2008/publication-6113.pdf |
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| Richardson, Tomas T.; Gilroy, Louise; Ishino, Yoshizumi; Connolly, Bernard A.; Henneke, Ghislaine. |
| Archaeal family-D DNA polymerase is inhibited by the presence of uracil in DNA template strands. When the enzyme encounters uracil, following three parameters change: DNA binding increases roughly 2-fold, the rate of polymerization slows by a factor of similar to 5 and 3'-5' proof-reading exonuclease activity is stimulated by a factor of similar to 2. Together these changes result in a significant decrease in polymerization activity and a reduction in net DNA synthesis. Pol D appears to interact with template strand uracil irrespective of its distance ahead of the replication fork. Polymerization does not stop at a defined location relative to uracil, rather a general decrease in DNA synthesis is observed. 'Trans' inhibition, the slowing of Pol D by uracil... |
| Tipo: Text |
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| Ano: 2013 |
URL: http://archimer.ifremer.fr/doc/00138/24975/23070.pdf |
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| Killelea, Tom; Ralec, Celine; Bosse, Audrey; Henneke, Ghislaine. |
| DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides,... |
| Tipo: Text |
Palavras-chave: DNA polymerase; Archaea; Family D; PCR; Pyrococcus. |
| Ano: 2014 |
URL: http://archimer.ifremer.fr/doc/00193/30450/28871.pdf |
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| 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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| Sauguet, Ludovic; Raia, Pierre; Henneke, Ghislaine; Delarue, Marc. |
| Archaeal replicative DNA polymerase D (PolD) constitute an atypical class of DNA polymerases made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2), both with unknown structures. We have determined the crystal structures of Pyrococcus abyssi DP1 and DP2 at 2.5 and 2.2 angstrom resolution, respectively, revealing a catalytic core strikingly different from all other known DNA polymerases (DNAPs). Rather, the PolD DP2 catalytic core has the same 'double-psi beta-barrel' architecture seen in the RNA polymerase (RNAP) superfamily, which includes multi-subunit transcriptases of all domains of life, homodimeric RNA-silencing pathway RNAPs and atypical viral RNAPs. This finding bridges together, in non-viral world, DNA... |
| Tipo: Text |
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| Ano: 2016 |
URL: http://archimer.ifremer.fr/doc/00350/46144/45842.pdf |
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| Madru, Clément; Henneke, Ghislaine; Raia, Pierre; Hugonneau-beaufet, Inès; Pehau-arnaudet, Gérard; England, Patrick; Lindahl, Erik; Delarue, Marc; Carroni, Marta; Sauguet, Ludovic. |
| Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD–PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach — combining cryo-EM, X-ray crystallography, protein–protein interaction measurements, and activity assays — we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second... |
| Tipo: Text |
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| Ano: 2020 |
URL: https://archimer.ifremer.fr/doc/00620/73180/72367.pdf |
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| Cannone, Giuseppe; Visentin, Silvia; Palud, Adeline; Henneke, Ghislaine; Spagnolo, Laura. |
| Cell division is a complex process that requires precise duplication of genetic material. Duplication is concerted by replisomes. The Minichromosome Maintenance (MCM) replicative helicase is a crucial component of replisomes. Eukaryotic and archaeal MCM proteins are highly conserved. In fact, archaeal MCMs are powerful tools for elucidating essential features of MCM function. However, while eukaryotic MCM2-7 is a heterocomplex made of different polypeptide chains, the MCM complexes of many Archaea form homohexamers from a single gene product. Moreover, some archaeal MCMs are polymorphic, and both hexameric and heptameric architectures have been reported for the same polypeptide. Here, we present the structure of the archaeal MCM helicase from Pyrococcus... |
| Tipo: Text |
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| Ano: 2017 |
URL: http://archimer.ifremer.fr/doc/00373/48434/48701.pdf |
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| Raia, Pierre; Carroni, Marta; Henry, Etienne; Pehau-arnaudet, Gerard; Brule, Sebastien; Beguin, Pierre; Henneke, Ghislaine; Lindahl, Erik; Delarue, Marc; Sauguet, Ludovic. |
| PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo–electron microscopy (cryo-EM) structure of the heterodimeric DP1–DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the... |
| Tipo: Text |
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| Ano: 2019 |
URL: https://archimer.ifremer.fr/doc/00477/58883/61420.pdf |
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| Le Laz, Sebastien; Le Goaziou, Audrey; Henneke, Ghislaine. |
| Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of Pyrococcus abyssi showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme. PabRNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA... |
| Tipo: Text |
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| Ano: 2010 |
URL: http://archimer.ifremer.fr/doc/00007/11834/8573.pdf |
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| 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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| 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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| Registros recuperados: 24 | |
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