Abstract
The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.
Highlights
The eukaryotic replisome is assembled and disassembled in a highly regulated fashion during each cell cycle, to ensure that each chromosome is duplicated just once (Bell & Labib, 2016; Burgers & Kunkel, 2017; Gasser, 2019)
Mouse ES cells represent an ideal model system with which to study the regulation of CMG disassembly in the mammalian cell cycle
Compared with diploid human cell lines such as RPE1, mouse ES cells have a number of features that facilitate studies of the CMG helicase at mammalian DNA replication forks
Summary
The eukaryotic replisome is assembled and disassembled in a highly regulated fashion during each cell cycle, to ensure that each chromosome is duplicated just once (Bell & Labib, 2016; Burgers & Kunkel, 2017; Gasser, 2019). During DNA replication termination, the key regulated step that induces CMG disassembly is ubiquitylation of the MCM7 subunit of the helicase (Maric et al, 2014; Moreno et al, 2014), which drives recruitment of the Cdc48 / p97 ATPase (Maric et al, 2017; Deegan et al, 2020). A similar reaction was observed when Xenopus egg extracts lacking CUL2LRR1 activity were driven into mitosis by premature activation of Cyclin-Dependent Kinase or CDK (Deng et al, 2019; Priego Moreno et al, 2019) In both worm and frog, mitotic CMG disassembly requires a metazoan-specific RING ubiquitin ligase known as TRUL-1 in C. elegans (TRAIP Ubiquitin Ligase 1) and TRAIP in vertebrates (Deng et al, 2019; Priego Moreno et al, 2019; Sonneville et al, 2019). We describe such a system based on mouse embryonic stem cells (mouse ES cells), revealing a conserved role in CMG disassembly for CUL2LRR1, TRAIP and p97
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