http://www.cnr.it/ontology/cnr/individuo/prodotto/ID9038
Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus. (Articolo in rivista)
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- Label
- Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus. (Articolo in rivista) (literal)
- Anno
- 2000-01-01T00:00:00+01:00 (literal)
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Pisani FM, De Felice M, Carpentieri F, Rossi M. (2000)
Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus.
in Journal of Molecular Biology
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- Pisani FM, De Felice M, Carpentieri F, Rossi M. (literal)
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- ISI Web of Science (WOS) (literal)
- Titolo
- Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus. (literal)
- Abstract
- Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoRFC is a hetero-pentamer composed of polypeptides of 37 kDa (small subunit) and 46 kDa (large subunit), which possess primary structure similarity with human replication factor C p40 and p140 subunits, respectively. The two SsoRFC polypeptides were co-expressed in Escherichia coli and purified as a complex (SsoRFC-complex) that was demonstrated to possess a native M(r) of about 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The small subunit was individually expressed in E. coli, purified, and found to form a homo-tetramer (SsoRFC-small; native M(r) 156 kDa), which was also characterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the synthetic activity of S. solfataricus B1-type DNA polymerase in reactions containing primed M13mp18 DNA, ATP, and either of the two poliferating cell nuclear antigen-like processivity factors of S. solfataricus (039p and 048p). Both SsoRFC-small and -complex were able to hydrolyze ATP, but only the ATPase activity of the holo-enzymatic assembly was activated by primed DNA templates, such as poly(dA)-oligo(dT). As measured by nitrocellulose filter binding assays, SsoRFC-complex bound poly(dA)-oligo(dT), but not the unprimed homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activity. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archaea are discussed. (literal)
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