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Title
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Biochemical analysis of replication factor C from the hyperthermophilic archaeon Pyrococcus furiosus.
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Authors
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I.K.Cann,
S.Ishino,
M.Yuasa,
H.Daiyasu,
H.Toh,
Y.Ishino.
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Ref.
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J Bacteriol, 2001,
183,
2614-2623.
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PubMed id
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Abstract
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Replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are
accessory proteins essential for processive DNA synthesis in the domain Eucarya.
The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA
polymerases delta and epsilon, onto primed DNA templates. RFC-like genes,
arranged in tandem in the Pyrococcus furiosus genome, were cloned and expressed
individually in Escherichia coli cells to determine their roles in DNA
synthesis. The P. furiosus RFC (PfuRFC) consists of a small subunit (RFCS) and a
large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which
was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA)
and P. furiosus PCNA (PfuPCNA). The ATPase activity 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 activity 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 polymerase I and polymerase II from P.
furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA
and that the role of RFC in processive DNA synthesis is conserved in Archaea and
Eucarya.
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