Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity
Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity
复制标题
DOI：
10.1074/jbc.273.10.5979
复制
发表时间：
1998-03-06
影响因子：
4.8
通讯作者：
Stillman, B
中科院分区：
文献类型：
--
作者：
Ellison, V;Stillman, B
复制因子C（replicationfactorC，RFC）是一种五亚基蛋白复合物，在真核细胞S期协调前导链和滞后链DNA的合成和DNA修复中起重要作用，它的功能是将增殖细胞核抗原（proliferatingcellnuclearantigen，PCNA）加载到引物DNA模板上，这一过程依赖于ATP，通过1）RFC识别引物末端（2）结合并破坏PCNA三聚体，然后3）将PCNA拓扑连接到DNA。在本报告中，我们描述了重组人RFC的纯化和性质表达在Sf 9细胞从杆状病毒表达载体，如天然RFC衍生自293细胞，重组RFC被发现支持SV 40 DNA合成和聚合酶δ DNA合成在体外，并具有ATP酶活性，高度刺激的DNA和PCNA进一步增强。观察到RFC的组装涉及不同的亚基相互作用，其中36-和38-kDa亚基与37-kDa亚基相互作用，并且40-kDa亚基与36-kDa亚基-37-kDa亚基亚复合物相互作用。发现140-kDa亚基主要需要与38-和40-kDa亚基相互作用以掺入复合物中。发现缺乏140-kDa亚基的稳定亚复合物，尽管其DNA复制有缺陷，但具有DNA依赖性ATP酶活性，其对加入PCNA无反应。
Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells, It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates, This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC (2) binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA. In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors, Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA. Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit sub complex, The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex, In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.