Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus

Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) ar e accessory proteins essential for processive DNA synthesis in the domain E ucarya. The function of RFC is to load PCNA, a processivity factor of eukar yotic DNA polymerases delta and epsilon, onto primed DNA templates. RFC-lik e genes, arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed individually in Escherichia call cells to determine their rol es in DNA synthesis. The P. furiosus RFC (PfuRFC) consists of a small subun it (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATP ase activity which was stimulated up to twofold in the presence of both sin gle-stranded DNA (ssDNA) and P. furiosus PCNA (PfuPCNA). The ATPase activit y of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activit y under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both pol ymerase I and polymerase II from P. furiosus. We propose that PfuRFC is req uired for efficient loading of PfuPCNA and that the role of RFC in processi ve DNA synthesis is conserved in Archaea and Eucarya.