Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.
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 (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.
Henneke
Ghislaine
G
Ifremer, Laboratoire de Microbiologie et Biotechnologie des Extrêmophiles, DRV/VP, BP 70, F-29280 Plouzané, France.
Gueguen
Yannick
Y
Flament
Didier
D
Azam
Philippe
P
Querellou
Joël
J
Dietrich
Jacques
J
Hübscher
Ulrich
U
Raffin
Jean-Paul
JP
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003429:Cross Reactions; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication; D019641:DNA, Archaeal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019766:Genes, Archaeal; D006801:Humans; D006868:Hydrolysis; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D019711:Pyrococcus / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C; D017386:Sequence Homology, Amino Acid
D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004257:DNA Polymerase II; D000251:Adenosine Triphosphatases; D051818:Replication Protein C