Binding to PCNA in Euryarchaeal DNA Replication Requires Two PIP Motifs for DNA Polymerase D and One PIP Motif for DNA Polymerase B

JOURNAL OF MOLECULAR BIOLOGY

卷:394

Article

Castrec, Benoit^1,2  Rouillon, Christophe^1,2  Henneke, Ghislaine^1,2  Flament, Didier^1,2  Querellou, Joel^1,2  Raffin, Jean-Paul^2,3 

[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

关键词: DNA replication;    DNA polymerases;    PCNA-binding motifs;    PIP box;    Archaea;   

DOI  :  10.1016/j.jmb.2009.09.044

来源: Elsevier

【 摘 要 】

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.

【 授权许可】

Free