Friedrich Miescher Institute for Biomedical Research, Basel, 4058, Switzerland.
University of Basel, Basel, 4051, Switzerland.
Nat Commun. 2022 Feb 25;13(1):1059. doi: 10.1038/s41467-022-28695-w.
The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5-2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.
在 DNA 复制之前，多个复制解旋酶加载因子的协调作用对于复制起点的许可（licensing）是必需的。原点识别复合物（ORC）与 DNA 的结合启动了 Cdc6、Cdt1 和 Mcm2-7 加载的 ATP 依赖性募集，但对于及时的 ATP 酶位点调节以及如何通过抑制信号（如细胞周期蛋白依赖性激酶磷酸化）来阻止加载的结构细节尚不清楚。我们使用冷冻电子显微镜，以 2.5-2.7Å 的分辨率确定了几种酿酒酵母 ORC·DNA·Cdc6 中间体的结构。这些结构揭示了起始子·共加载器组装的不同环构象和 ORC 和 Cdc6 的无活性 ATP 酶位点构型。Orc6 N 端结构域以一种与有效 Mcm2-7 对接不兼容的方式侧向结合 ORC·Cdc6 环，而删除该 Orc6 区域可减轻 CDK 介导的 Mcm7 募集抑制。我们的研究结果支持了一种模型，即 Orc6 促进了自动抑制的 ORC·DNA·Cdc6 中间体的组装，以响应 CDK 磷酸化来阻止原点许可，并避免 DNA 再复制。
