Binding to PCNA in Euryarchaeal DNA Replication Requires Two PIP Motifs for DNA Polymerase D and One PIP Motif for DNA Polymerase B
|Author(s)||Castrec Benoit1, 2, Rouillon Christophe1, 2, Henneke Ghislaine1, 2, Flament Didier1, 2, Querellou Joel1, 2, Raffin Jean-Paul2, 3|
|Affiliation(s)||1 : Univ Bretagne Occidentale, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
2 : IFREMER, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
3 : CNRS, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
|Source||Journal of Molecular Biology (0022-2836) (Elsevier), 2009-11 , Vol. 394 , N. 2 , P. 209-218|
|WOS© Times Cited||23|
|Keyword(s)||Archaea, PIP box, PCNA binding motifs, DNA polymerases, DNA replication|
|Abstract||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 D with PCNA. Thus, two PIP motifs were needed for PabPol D for binding to PabPCNA. Moreover, the C-terminus of PabPCNA was essential for stimulation of PabPol D activity but not for stimulation of PabPol B activity. Neither DNA polymerase interacted with the PabPCNA interdomain connecting loop. Our data suggest that distinct processes are involved in PabPol D and PabPol B binding to PCNA, raising the possibility that Archaea require two mechanisms for recruiting replicative DNA polymerases at the replication fork. (C) 2009 Elsevier Ltd. All rights reserved.|