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| 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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| 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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| 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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| Henneke, Ghislaine; Raffin, Jean-paul; Ferrari, Elena; Jonsson, Zophonías; Dietrich, Jacques; Hubscher, Ulrich. |
| We have cloned the gene encoding proliferating cell nuclear antigen (PCNA) from the hyperthermophilic euryarchaeote Thermococcus fumicolans (Tfu). Tfu PCNA contains 250 amino acids with a calculated M-r of 28,000 and is 26% identical to human PCNA. Next, Tfu PCNA was overexpressed in Escherichia coli and it showed an apparent molecular mass of 33.5 kDa. The purified Tfu PCNA was tested first with recombinant Tfu DNA polymerase I (Tfu pol) and second with calf thymus DNA polymerase delta (pol delta). When tested with the homologous Tfu pol on bacteriophage lambda DNA, large amounts of Tfu PCNA were required to obtain two- to threefold stimulation. Surprisingly, however, Tfu PCNA was much more efficient than human PCNA in stimulating calf thymus pol delta.... |
| Tipo: Text |
Palavras-chave: DNA polymerase delta; Conservation; Thermococcus fumicolans; Archaea; Hyperthermophile; PCNA. |
| Ano: 2000 |
URL: http://archimer.ifremer.fr/doc/2000/publication-659.pdf |
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