Abstract
Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45–MCM–GINS (CMG) replicative helicase1–3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4–7), 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 DNA8–10. However, it is unknown how SCFDia2 and CUL2LRR1 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.
Highlights
Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia[2] in budding yeast[1], CUL2LRR1 in metazoa4–7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse
The replisome is disassembled in two steps: first, CMG is ubiquitylated on its Mcm[7] subunit by a cullin-RING E3 ubiquitin ligase (SCFDia[2] in budding yeast, CUL2LRR1 in metazoa); second, ubiquitylated Mcm[7] is unfolded by the Cdc[48] ATPase, leading to disassembly of the replisome[1,2,3,8,9,10]
To trap a replisome bound around double-stranded DNA (dsDNA), we used a DNA substrate that lacked a 5′ flap and contained a short stretch methylphosphonate modifications embedded in dsDNA, which slow translocation of CMG15 (Extended Data Fig. 1a)
Summary
A Conserved Mechanism for Regulating Replisome Disassembly in Eukaryotes Jenkyn-Bedford, Michael; Jones, Morgan L.; Baris, Yasemin; Labib, Karim P. Citation for published version (APA): Jenkyn-Bedford, M., Jones, M. A Conserved Mechanism for Regulating Replisome Disassembly in Eukaryotes.
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