Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus

Here we report the isolation and characterization of a clamp-loader complex from the thermoacidophilic archaeon Sulfolobus solfataricus (SsoRFC). SsoR FC 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 Ss oRFC polypeptides were co-expressed in Escherichia coli and purified as a c omplex (SsoRFC-complex) that was demonstrated to possess a native M-r of ab out 200 kDa and a 4:1 (small to large) subunit stoichiometric ratio. The sm all subunit was individually expressed in E. coli, purified,and found to fo rm a homo-tetramer (SsoRFC-small; native M-r 156 kDa), which was also chara cterized. The SsoRFC-complex, but not SsoRFC-small, highly stimulated the s ynthetic activity of S. solfataruicus B1-type DNA polymerase in reactions c ontaining 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 unpri med homopolymer, whereas SsoRFC-small was devoid of any DNA-binding activit y. The peculiar properties of this archaeal clamp-loader complex and their significance for the understanding of the DNA replication process in Archae a are discussed. (C) 2000 Academic Press.