Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork
Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork
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DOI：
10.1016/j.molcel.2020.04.012
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发表时间：
2020-06-04
期刊：
影响因子：
16
通讯作者：
Yeeles, Joseph T. P.
中科院分区：
文献类型：
--
作者：
Baretic, Domagoj;Jenkyn-Bedford, Michael;Yeeles, Joseph T. P.
真核复制体围绕cdc45-MCM-gins(CMG)解旋酶组织，协调染色体复制。多种因素直接与CMG相关，包括Ctf4和异三聚体保护复合体(Csm3/Tof1和MRc1)，它具有重要的作用，包括帮助正常复制速度和稳定失速的叉子。这些蛋白质是如何与CMG结合来执行这些功能的，目前还知之甚少。在这里，我们提出了3到3.5埃分辨率的电子冷冻显微镜(CRYO-EM)结构，包括CMG、Ctf4和复制叉处的叉子保护复合体。这些结构提供了CMG-DNA相互作用的高分辨率视图，揭示了链分离的机制，并通过对高效复制分叉暂停至关重要的相互作用网络显示Csm3/Tof1“GRIP”双链DNA领先于CMG。尽管在我们的结构中没有解析MRc1，但我们通过交联质谱确定了其在复制体中的拓扑结构。我们的工作共同揭示了四个高度保守的复制体组件如何与CMG合作来促进复制体的进展和维持基因组的稳定。
The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG, including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), which has important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3 to 3.5 angstrom resolution electron cryomicroscopy (cryo-EM) structures comprising CMG, Ctf4, and the fork protection complex at a replication fork. The structures provide high-resolution views of CMG-DNA interactions, revealing a mechanism for strand separation, and show Csm3/Tof1 "grip'' duplex DNA ahead of CMG via a network of interactions important for efficient replication fork pausing. Although Mrc1 was not resolved in our structures, we determine its topology in the replisome by cross-linking mass spectrometry. Collectively, our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.