Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells
Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells
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DOI：
10.1073/pnas.93.23.12896
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发表时间：
1996-11-12
影响因子：
11.1
通讯作者：
Hurwitz, J
中科院分区：
文献类型：
--
作者：
Cai, JS;Uhlmann, F;Hurwitz, J
人复制因子C （RFC，也称为激活因子I）是一种五亚基蛋白复合物（p140, p10, p38， p37和p36），是增殖细胞核抗原（PCNA）依赖的DNA合成所必需的，由DNA聚合酶delta或epsilon催化。在这里，我们报道了在杆状病毒感染的昆虫细胞中同时过表达的RFC复合体的五个亚基的重建。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，并被证明在功能上与其天然对偶物相同(i)支持DNA聚合酶催化的DNA依赖链延伸；（ii）催化由PCNA和人单链DNA结合蛋白刺激的DNA依赖性ATP水解；（iii）优先结合DNA引物末端；（iv）催化将PCNA装载到单缺口的环状DNA上，并催化从这些DNA分子中去除PCNA。
Human replication factor C (RFC, also called activator I) is a five-subunit protein complex (p140, p10, 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 comples 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 preferentiaIly to DNA primer ends; and (iv) catalytically loading PCNA onto singly nicked circular DNA and catalytically removing PCNA from these DNA molecules.