Macromolecular Machines Laboratory, Francis Crick Institute, London, UK.
Chromosome Replication Laboratory, Francis Crick Institute, London, UK.
Nature. 2019 Nov;575(7784):704-710. doi: 10.1038/s41586-019-1768-0. Epub 2019 Nov 20.
In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA. The mechanism of MCM double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
为了准备双向 DNA 复制，起始识别复合物 (ORC) 将两个六聚体 MCM 解旋酶加载到 DNA 周围形成对头双六聚体。MCM 双六聚体形成的机制存在争议。单分子实验表明存在顺序机制，其中 ORC 依赖性加载第一个六聚体驱动第二个六聚体的募集。相比之下，生化数据表明，两个环通过相同的 ORC 介导的机制独立加载，在两个反向 DNA 位点。在这里，我们使用时间分辨电子显微镜可视化 MCM 加载，并鉴定双六聚体形成中的中间体。我们证实两个六聚体都是通过 ORC 与 MCM 解旋酶 C 端结构域之间发生的相同相互作用募集的。此外，我们确定了耦合 MCM 加载的机制。第一个 MCM 六聚体在 DNA 周围的加载创建了一个独特的相互作用位点，该位点促进了 ORC 在 MCM N 端同源二聚化界面的结合。在此构型中，ORC 准备以倒置取向指导第二个六聚体的募集，这适合双六聚体的形成。因此，我们的结果调和了源自单分子实验和生化数据的两个明显矛盾的模型。
