Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus
Fm. Pisani et al., Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus, J MOL BIOL, 301(1), 2000, pp. 61-73
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.