Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome
Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome
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DOI：
10.1016/j.celrep.2019.07.104
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发表时间：
2019-09-03
期刊：
影响因子：
8.8
通讯作者：
Costa, Alessandro
中科院分区：
文献类型：
--
作者：
Eickhoff, Patrik;Kose, Hazal B.;Costa, Alessandro
在真核复制体中，Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) 解旋酶解旋 DNA 需要一种名为微型染色体维持 (MCM) 的六聚环状 ATP 酶，该酶通过其中央通道卷绕单链 DNA。并非所有六个 ATP 酶位点都是解旋所必需的；然而，解旋酶机制尚不清楚。我们使用冷冻电子显微镜 (cryo-EM) 对 ATP 水解驱动的 CMG 易位进行成像，发现六个 MCM 亚基一次使用四个相邻原聚体与 DNA 结合，ATP 结合促进 DNA 结合。不同解旋酶状态之间的变形使我们提出了一种非对称的交手旋转机制，解释了 CMG 的 MCM 环周围 ATP 酶功能的不对称要求。通过对高阶复制体组装的成像，我们发现Mrc1-Csm3-Tof1叉稳定复合物增强了亲本双链DNA与叉处CMG之间的相互作用，这可能支持DNA易位和叉解旋之间的耦合。
In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.