Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6
Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6
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DOI：
10.1073/pnas.2006231117
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发表时间：
2020-07-28
影响因子：
11.1
通讯作者：
Speck, Christian
中科院分区：
文献类型：
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作者：
Yuan, Zuanning;Schneider, Sarah;Speck, Christian
DNA复制起点作为复制解旋酶装载的位点。在所有真核生物中，六亚基起始识别复合体（Orc1-6； ORC）识别复制起始。在细胞周期的m期后期，Cdc6与ORC结合，ORC-Cdc6复合物在多步反应中装载，并在Cdt1的帮助下，将核心Mcm2-7解旋酶装载到DNA上。一个关键的中间体是ORC-Cdc6-Cdt1-Mcm2-7 （OCCM）复合体，其中DNA已经插入到Mcm2-7的中心通道中。到目前为止，人们还不清楚起源DNA是如何被ORC-Cdc6引导并插入到Mcm2-7六聚体中的。在这里，我们截断Mcm6的c端翼螺旋结构域（WHD）以减缓加载反应，从而在DNA插入芽殖酵母之前捕获两个加载中间体。在“半连接OCCM”中，Mcm3和Mcm7 whd锁存到ORC-Cdc6上，而Mcm2-7六聚体的主体没有连接。在“预插入OCCM”中，Mcm2-7的主体停靠在ORC-Cdc6上，起源DNA弯曲并定位在开放的DNA入口门附近，在Mcm2-Mcm5界面上准备插入。我们利用分子模拟揭示了从预加载构象到加载构象的动态转变，其中Mcm2-7在DNA上的加载完成并且DNA入口门完全关闭。我们的工作为真核生物DNA复制的关键事件提供了多个分子见解。
DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition com-plex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from pre -loading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.