CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold.

Tom D Deegan,Karim Labib,Ryo Fujisawa,Cristian Polo Rivera,Progya P Mukherjee

doi:10.7554/elife.60371

Tom D Deegan, Karim Labib + Show 3 more

Open Access

https://doi.org/10.7554/elife.60371

Copy DOI

Journal: eLife	Publication Date: Aug 17, 2020
Citations: 55	License type: CC BY 4.0

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit

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.

Highlights

Eukaryotic chromosomes are duplicated just once per cell cycle, by a large molecular machine called the replisome (Bell and Labib, 2016; Burgers and Kunkel, 2017)
Replisome assembly is initiated in the G1-phase of the cell cycle, when the six Mcm2-7 ATPases are loaded around double strand DNA at origins of replication, as a ‘head-to-head’ double hexamer (Evrin et al, 2009; Li et al, 2015; Remus et al, 2009)
DNA replication termination is defective when two forks converge in this system, unless the reactions contain one of the two yeast members of the Pif1 helicase family, which help the replisome to unwind the final stretch of parental DNA (Deegan et al, 2019)

Summary

Introduction

Eukaryotic chromosomes are duplicated just once per cell cycle, by a large molecular machine called the replisome (Bell and Labib, 2016; Burgers and Kunkel, 2017). The transient opening of a poorly defined ‘gate’ in CMG leads to complete exclusion of the lagging strand DNA template from the Mcm central channel (Wasserman et al, 2019), in a step that requires the Mcm protein (Douglas et al, 2018). This produces two activated CMG helicases that encircle opposite strands of the parental DNA duplex.

Methods

Results

Conclusion