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Nucleic Acids Research, 2002, Vol. 30, No. 20 4329-4338
© 2002 Oxford University Press

Biochemical characterisation of the clamp/clamp loader proteins from the euryarchaeon Archaeoglobus fulgidus

Anja Seybert, David J. Scott1, Sarah Scaife, Martin R. Singleton and Dale B. Wigley*

Molecular Enzymology Laboratory, London Research Institute, Clare Hall Laboratories, Cancer Research UK, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK and 1 Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 1TD, UK

*To whom correspondence should be addressed. Tel: +44 207 269 3930; Fax: +44 207 269 3803; Email: dale.wigley{at}cancer.org.uk

Replicative polymerases of eukaryotes, prokaryotes and archaea obtain processivity using ring-shaped DNA sliding clamps that are loaded onto DNA by clamp loaders [replication factor C (RFC) in eukaryotes]. In this study, we cloned the two genes for the subunits of the RFC homologue of the euryarchaeon Archaeoglobus fulgidus. The proteins were expressed and purified from Escherichia coli both individually and as a complex. The afRFC subunits form a heteropentameric complex consisting of one copy of the large subunit and four copies of the small subunits. To analyse the functionality of afRFC, we also expressed the A.fulgidus PCNA homologue and a type B polymerase (PolB1) in E.coli. In primer extension assays, afRFC stimulated the processivity of afPolB1 in afPCNA-dependent reactions. Although the afRFC complex showed significant DNA-dependent ATPase activity, which could be further stimulated by afPCNA, neither of the isolated afRFC subunits showed this activity. However, both the large and small afRFC subunits showed interaction with afPCNA. Furthermore, we demonstrate that ATP binding, but not hydrolysis, is needed to stimulate interactions of the afRFC complex with afPCNA and DNA.


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