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

JOURNAL OF MOLECULAR BIOLOGY

卷:323

Article

Henneke, G ; Gueguen, Y ; Flament, D ; Azam, P ; Querellou, J ; Dietrich, J ; Hübscher, U ; Raffin, JP

关键词: replication factor C;    archaea;    hyperthermophile;    Pyrococcus abyssi;    PCNA-binding domain;   

DOI  :  10.1016/S0022-2836(02)01028-8

来源: Elsevier

【 摘 要 】

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 (Pab RFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the Pab RFC-small subunit could be purified, while the large subunit (Pab RFC-large) alone was completely insoluble. The highly purified Pab RFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the Pab RFC complex in a DNA-dependent manner, but the Pab RFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The Pab RFC complex was able to stimulate Pab PCNA-dependent DNA synthesis by the Pab-family D heterodimeric DNA polymerase. Finally, (i) the Pab RFC-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 Pab RFC complex ATPase activity in a DNA-dependent way and (iii) the Pab RFC 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. (C) 2002 Elsevier Science Ltd. All rights reserved.

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