Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms EN6 3LD, UK.
Nature. 2013 Mar 21;495(7441):339-43. doi: 10.1038/nature11920. Epub 2013 Mar 10.
The regulated loading of the Mcm2-7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2-7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin 'licensing' is still poorly understood. Here we show that both Mcm2-7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC-Cdc6. ATP hydrolysis can promote Mcm2-7 loading, but can also promote Mcm2-7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication.
Mcm2-7 DNA 解旋酶（由六个相关亚基，Mcm2 到 Mcm7 组成）在多个复制起点被调控装入复制前复合物，以确保真核细胞中精确的每个细胞周期复制一次。起始识别复合物（ORC）、Cdc6 和 Cdt1 将 Mcm2-7 加载到一个双六聚体中，该六聚体结合在双链 DNA 周围，这是一个依赖 ATP 的反应，但这种起始“许可”的分子机制仍知之甚少。在这里，我们表明酵母中的 Mcm2-7 六聚体都被 Mcm3 的必需保守羧基末端结构域招募到起始点，该结构域与 ORC-Cdc6 相互作用并刺激其 ATP 酶活性。ATP 水解可以促进 Mcm2-7 的加载，但如果缺少组件或 ORC 已被细胞周期蛋白依赖性激酶磷酸化失活，ATP 水解也可以促进 Mcm2-7 的释放。我们的工作提供了对起始点如何被许可的新见解，并揭示了一种新的 ATP 酶依赖性机制，有助于精确的每个细胞周期复制。
