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首页> 外文期刊>Journal of Molecular Biology >Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus.
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Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solfataricus.

机译：与来自超嗜热古细菌Sulfolobus solfataricus的真核复制因子C同源的钳-装载物复合物的生化特征。

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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. Copyright 2000 Academic Press.
机译：在这里，我们报告了嗜热古细菌Sulfolobus solfataricus（SsoRFC）的钳夹加载物复合物的分离和表征。 SsoRFC是由37 kDa（小亚基）和46 kDa（大亚基）多肽组成的杂戊烷，它们分别与人复制因子C p40和p140亚基具有一级结构相似性。两种SsoRFC多肽在大肠杆菌中共表达，并纯化为复合物（SsoRFC-复合物），该复合物被证明具有约200 kDa的天然M（r）和4：1（小到大）的亚单位化学计量比。该小亚基在大肠杆菌中单独表达，纯化并发现形成同型四聚体（SsoRFC-small;天然M（r）156 kDa），并对其进行了表征。在包含引发的M13mp18 DNA，ATP和S的两个增殖性细胞核抗原样增生性因子之一的反应中，SsoRFC复杂而不是SsoRFC很小的混合物极大地刺激了S. solfataricus B1型DNA聚合酶的合成活性。 solfataricus（039p和048p）。 SsoRFC-small和-complex都能够水解ATP，但是只有带底漆的DNA模板（例如poly（dA）-oligo（dT））才激活完整酶促组装的ATPase活性。如通过硝酸纤维素滤膜结合测定法所测量，SsoRFC-复合物结合了poly（dA）-oligo（dT），但未结合的未涂底均聚物，而SsoRFC-small没有任何DNA结合活性。讨论了这种古细菌钳-装载物复合物的独特特性及其对理解古细菌DNA复制过程的意义。版权所有2000学术出版社。

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