Binding to PCNA in Euryarchaeal DNA Replication requires two PIP motifs for DNA polymerase D and one PIP motif for DNA polymerase B.
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.
Université de Bretagne Occidentale, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes, BP 70, F-29280 Plouzané, France.
Benoît
B
Castrec
Christophe
C
Rouillon
Ghislaine
G
Henneke
Didier
D
Flament
Joël
J
Querellou
Jean-Paul
JP
Raffin
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004257:DNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D043903:Pyrococcus abyssi / Q000235:genetics / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase