ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits
ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits
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DOI：
10.1073/pnas.95.20.11607
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发表时间：
1998-09-29
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;Yao, NN;Hurwitz, J
人类复制因子 C (hRFC) 是一种五亚基蛋白质复合物（p140、p40、p38、p37 和 p36），其作用是将增殖细胞核抗原催化加载到 DNA 上，以消耗 ATP 的方式将 DNA 聚合酶 δ 或 ε 募集到引物末端，从而导致持续的 DNA 合成。我们之前已经证明，由三个亚基（p40、p37 和 p36）组成的 hRFC 子复合体包含 DNA 依赖性 ATP 酶活性。然而，尚不清楚哪个亚基水解 ATP，因为所有五个亚基都包含潜在的 ATP 结合位点。在本报告中，我们在每个 hRFC 亚基的假定 ATP 结合序列中引入了点突变，并检查了所得突变 hRFC 复合物的特性以及 hRFC 或 p40 p37 p36 复合物的 ATP 酶活性。 p36、p37、p40 或 p140 亚基的 ATP 结合位点之一的突变显着降低 hRFC 复合物的复制活性以及 hRFC 或 p40 p37 p36 复合物的 ATP 酶活性。 p38 亚基的 ATP 结合位点的突变不会改变 hRFC 的复制活性。这些发现表明 hRFC 的复制活性依赖于四个 hRFC 亚基的作用所促成的有效 ATP 水解。
Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report,,ve introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40 p37 p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40 p37 p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC, These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.