Biochemical characterisation of the clamp/clamp loader proteins from the euryarchaeon Archaeoglobus fulgidus.
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.
Seybert
Anja
A
Molecular Enzymology Laboratory, London Research Institute, Clare Hall Laboratories, Cancer Research UK.
Scott
David J
DJ
Scaife
Sarah
S
Singleton
Martin R
MR
Wigley
Dale B
DB
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019611:Archaeoglobus fulgidus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D019766:Genes, Archaeal; D046911:Macromolecular Substances; D008970:Molecular Weight; D011119:Polynucleotides / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D021122:Protein Subunits; D012313:RNA / Q000378:metabolism; D051818:Replication Protein C; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D011119:Polynucleotides; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D051818:Replication Protein C