CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold
- University of Dundee, United Kingdom
Abstract
The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48 / p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.
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All data generated or analysed during this study are included in the manuscript and supporting files.
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Author details
Funding
Medical Research Council (MC_UU_12016/13)
- Tom D Deegan
- Pragya P Mukherjee
- Ryo Fujisawa
- Cristian Polo Rivera
- Karim Labib
Wellcome (102943/Z/13/Z)
- Karim Labib
Wellcome (204678/Z/16/Z)
- Tom D Deegan
Cancer Research UK (C578/A24558)
- Ryo Fujisawa
- Karim Labib
Cancer Research UK (C578/A25669)
- Cristian Polo Rivera
- Karim Labib
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Deegan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold
eLife 9:e60371.
https://doi.org/10.7554/eLife.60371
