ATP bound to the origin recognition complex is important for preRC formation
ATP bound to the origin recognition complex is important for preRC formation
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DOI：
10.1073/pnas.131006898
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发表时间：
2001-07-17
影响因子：
11.1
通讯作者：
Bell, SP
中科院分区：
文献类型：
--
作者：
Klemm, RD;Bell, SP
起点识别复合物（ORC）结合复制起点并指导在这些位点组装更高级的蛋白质复合物。 ORC 在体外结合并水解 ATP。 ATP 与 ORC 最大亚基 Orc1p 结合，刺激与原始 DNA 的特异性结合；然而，ORC 水解 ATP 的功能尚不清楚。为了解决 ATP 水解的作用，我们在 Orc1p 中生成了显性致死突变体。在生理 ATP 浓度下，这些突变体在缺乏 DNA 的情况下，ATP 水解有缺陷，但没有 ATP 结合缺陷。这些突变体在过表达时会抑制复制前复合物的形成。这些突变型 ORC 复合物的显性致死表型受到野生型（而非突变型）Cdc6p 同时过表达的抑制。我们的研究结果表明，这些水解缺陷突变体通过滴定 Cdc6p 使其远离原点来抑制生长。基于这些观察，我们提出 Cdc6p 特异性识别 Orc1p 的 ATP 结合状态，并且 ATP 水解与 preRC 分解耦合。
The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA, These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based an these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.