Macromolecular Machines Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
Single Molecule Imaging of Genome Duplication and Maintenance Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
Cell Rep. 2019 Sep 3;28(10):2673-2688.e8. doi: 10.1016/j.celrep.2019.07.104.
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.
在真核复制体中，Cdc45-MCM-Go-Ichi-Ni-San（GINS）（CMG）解旋酶通过六聚体环形 ATP 酶解旋需要一种称为微小染色体维持（MCM）的六聚体环形 ATP 酶，该酶通过其中心通道缠绕单链 DNA。并非所有六个 ATP 酶位点都需要解旋；然而，解旋酶的机制尚不清楚。我们使用冷冻电镜（cryo-EM）成像 ATP 水解驱动的 CMG 易位，发现六个 MCM 亚基一次使用四个相邻的原聚体与 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手拉手旋转机制，解释了 CMG 中 MCM 环周围 ATP 酶功能的不对称要求。通过对更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偶联。
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