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EMBO Rep
2021 Mar 03;223:e52164. doi: 10.15252/embr.202052164.
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CUL2LRR1 , TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle.
Villa F, Fujisawa R, Ainsworth J, Nishimura K, Lie-A-Ling M, Lacaud G, Labib KP.
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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.
C578/A25669 Cancer Research UK (CRUK), JP20K21423 MEXT | Japan Society for the Promotion of Science (JSPS), MC_UU_12016/13 UKRI | Medical Research Council (MRC), BB/I001794/1/BB Biotechnology and Biological Sciences Research Council , MC_UU_00018/4 Medical Research Council , JP19K06611 MEXT | Japan Society for the Promotion of Science (JSPS), Wellcome Trust , C5759/A20971 Cancer Research UK (CRUK), Naito Foundation (), C578/A24558 Cancer Research UK (CRUK), 102943/Z/13/Z Wellcome Trust (WT), C578/A25669 Cancer Research UK, MC_UU_12016/13 Medical Research Council , C5759/A20971 Cancer Research UK, C578/A24558 Cancer Research UK, 102943/Z/13/Z Wellcome Trust
Figure 1. Mouse ES cells provide a model system for studying the mammalian CMG helicase
A. B. C. D. E. F. G. H. I. J.
Figure EV1. GINS co‐purifies with a range of replisome components from homozygous TAP‐SLD5 mouse ES cellsCell extracts from mouse ES lines with the indicated genotypes were incubated with IgG beads. The isolated proteins were analysed by immunoblotting in order to monitor the factors that co‐purified with TAP‐SLD5. Note that isolation of the CMG was more effective from homozygous TAP‐SLD5 / TAP‐SLD5 cells than from the equivalent heterozygote
Figure 2. The p97 ATPase is required for chromatin extraction and disassembly of ubiquitylated CMG helicase in mouse ES cells
A. B. C. D. E.
Figure EV2. Ubiquitylation of CMG‐MCM7 by CUL2LRR1
A. B. C.
Figure EV3. Accumulation of replisome components on chromatin in response to p97 inhibition
A. B. C. D. E.
Figure 3. CUL2LRR1 is required for CMG ubiquitylation and replisome disassembly during S‐phase in mouse ES cells
A. B. C. D. E.
Figure 4. Upon inhibition of CUL2LRR1, CMG persists on chromatin throughout interphase and is removed during early mitosis
A. B. C.
Figure 5. Mitotic CMG disassembly in mouse ES cells is dependent upon TRAIP E3 ligase activity but does not require association of TRAIP with PCNA
A. B. C. D.
Figure EV4. TRAIP−/− mouse ES cells are viable but are sensitive to DNA damaging agents that induce inter‐strand DNA crosslinks
A. B. C. D. E. F. G. H.
Figure EV5. Models for disassembly of the metazoan replisome. The figure summarises data from this manuscript together with previous studies with early embryos of Caenorhabditis elegans and extracts of Xenopus laevis eggs
A. B. C.
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