Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45–MCM–GINS (CMG) replicative helicase^1,2,3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF^Dia2 in budding yeast¹, CUL2^LRR1 in metazoa^4,5,6,7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA^8,9,10. However, it is unknown how SCF^Dia2 and CUL2^LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome–E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR–MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

复制体分解是真核 DNA 复制的最后一步，由 CDC45–MCM–GINS (CMG) 复制解旋酶的泛素化触发^1,2,3 。尽管由不同真核生物中进化多样的 E3 泛素连接酶驱动（芽殖酵母中的 SCF ^Dia2 ，^后生动物中的 CUL2 ^{LRR1 4,5,6,7} ），复制体分解受到共同的调节原则的控制，其中 CMG 的泛素化受到抑制在复制终止之前，以防止复制叉崩溃。最近的证据表明这种抑制是由复制叉 DNA 介导的^8,9,10 。然而，尚不清楚 SCF ^Dia2和 CUL2 ^LRR1如何区分终止延长复制体，从而仅在终止后选择性地泛素化 CMG。在这里，我们使用冷冻电子显微镜来解析出芽酵母和人类复制体-E3 连接酶组件的高分辨率结构。我们的结构表明，Dia2 和 LRR1 的富含亮氨酸重复结构域在结构上不同，但与 CMG 上的共同位点结合，包括 MCM3 和 MCM5 锌指结构域。 LRR-MCM 相互作用对于复制体分解至关重要，最重要的是，它被复制叉处排除的 DNA 链封闭，从而为终止前抑制 CMG 泛素化奠定了结构基础。我们的结果阐明了真核生物中复制体分解调节的保守机制，并揭示了 DNA 在保持复制体完整性方面先前未预料到的作用。