The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45–MCM–GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown¹. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.

真核复制起点的激活发生在时间上分开的步骤中，以确保每个细胞周期仅复制一次染色体。首先，MCM 解旋酶作为无活性的双六聚体加载到双链 DNA 上。在募集了一组组装两个 Cdc45-MCM-GINS (CMG) 全息解旋酶的激发因子后会发生激活。CMG 形成导致 DNA 在建立复制叉的路径上卷曲，但 DNA 是否在这个阶段熔化尚不清楚¹. 在这里，我们使用冷冻电子显微镜对染色质原点上的 ATP 依赖性 CMG 组装进行成像，并在体外用纯化的酵母蛋白重组。我们发现 CMG 的形成破坏了双六聚体界面，从而暴露了两个 CMG 之间的双链 DNA。这两个解旋酶保持束缚，从而产生展开的二聚体，对起源激活和复制体完整性有影响。在每个 MCM 环内，双螺旋变得松散并且碱基配对被破坏。这是 ATP 触发的 MCM 构象变化的结果，涉及 DNA 拉伸和蛋白质介导的三个孤儿碱基的稳定化。在我们的结构中接合 DNA 的 Mcm2 孔环残基对于双六聚体加载和 CMG 形成是可有可无的，但对于解开 DNA 和促进复制是必不可少的。