Cai J, Uhlmann F, Gibbs E, Flores-Rozas H, Lee C G, Phillips B, Finkelstein J, Yao N, O'Donnell M, Hurwitz J
Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12896-901. doi: 10.1073/pnas.93.23.12896.
Human replication factor C (RFC, also called activator 1) is a five-subunit protein complex (p140, p40, p38, p37, and p36) required for proliferating cell nuclear antigen (PCNA)-dependent processive DNA synthesis catalyzed by DNA polymerase delta or epsilon. Here we report the reconstitution of the RFC complex from its five subunits simultaneously overexpressed in baculovirus-infected insect cells. The purified baculovirus-produced RFC appears to contain equimolar levels of each subunit and was shown to be functionally identical to its native counterpart in (i) supporting DNA polymerase delta-catalyzed PCNA-dependent DNA chain elongation; (ii) catalyzing DNA-dependent ATP hydrolysis that was stimulated by PCNA and human single-stranded DNA binding protein; (iii) binding preferentially to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.
人类复制因子C（RFC，也称为激活因子1）是一种由五个亚基组成的蛋白质复合物（p140、p40、p38、p37和p36），它是DNA聚合酶δ或ε催化的依赖增殖细胞核抗原（PCNA）的连续DNA合成所必需的。在此，我们报告了从在杆状病毒感染的昆虫细胞中同时过表达的五个亚基重建RFC复合物。纯化的杆状病毒产生的RFC似乎含有等摩尔水平的每个亚基，并且在以下方面显示出与其天然对应物功能相同：（i）支持DNA聚合酶δ催化的依赖PCNA的DNA链延伸；（ii）催化由PCNA和人类单链DNA结合蛋白刺激的依赖DNA的ATP水解；（iii）优先结合DNA引物末端；以及（iv）将PCNA催化加载到单切口环状DNA上并从这些DNA分子上催化去除PCNA。
