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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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Les ADN polymérases B et D de l'Archaea hyperthermophile, Pyrococcus abyssi : contribution à l'étude des relations structure-fonction ArchiMer
Castrec, Benoit.
DNA replication is a functionally conserved mechanism among the three domains of life. This processus is performed by DNA polymerases with two accessories factors, PCNA and RF-C, sliding clamp and clamp loader, respectively. Our model of study, the Euryarchaeota Pyrococcus abyssi, possesses one monomeric DNA polymerase, belonging to the Family B, and one heterodimeric DNA polymerase, belonging to the Family D. Usually, proteins interact with PCNA at a hydrophobic pocket which is formed by the IDCL (interdomain connecting loop) and the C-terminus. These interactions are mediated through common motifs like the PIP box (motif PCNA interacting Protein). We have demonstrated that Pol B has just one PIP box motif at the C-terminus, while Pol D has two PIP-type...
Tipo: Text Palavras-chave: Thermostability; Interactions; Motifs; DNA replication; RF C; PCNA; DNA polymerases; Archaea; Thermostabilité; Interactions; Motifs; Réplication de l'ADN; RF C; PCNA; ADN polymérases; Archaea.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/these-6940.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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