In Vitro Reconstitution Defines the Minimal Requirements for Cdc48-Dependent Disassembly of the CMG Helicase in Budding Yeast

Overview

Journal Cell Rep

Publisher Cell Press

Specialties Biology
Cell Biology
Molecular Biology

Date 2019 Sep 12

PMID 31509741

Citations 15

Authors

Progya P Mukherjee

Karim P M Labib

Affiliations

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Abstract

Disassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCF. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of replisome disassembly that are observed in vivo. Our findings define the minimal requirements for disassembly of the eukaryotic replisome and provide a model system for studying the disassembly of protein complexes by Cdc48-Ufd1-Npl4.

Citing Articles

CMG helicase disassembly is essential and driven by two pathways in budding yeast.

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Structural basis of ubiquitin-independent PP1 complex disassembly by p97.

Boom J, Marini G, Meyer H, Saibil H EMBO J. 2023; 42(14):e113110.

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Targeting of client proteins to the VCP/p97/Cdc48 unfolding machine.

Meyer H, Boom J Front Mol Biosci. 2023; 10:1142989.

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Cooperative assembly of p97 complexes involved in replication termination.

Kochenova O, Mukkavalli S, Raman M, Walter J Nat Commun. 2022; 13(1):6591.

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