CUL-2LRR-1 and UBXN-3 drive replisome disassembly during DNA replication termination and mitosis

Abstract

Replisome disassembly is the final step of DNA replication in eukaryotes, involving the ubiquitylation and CDC48-dependent dissolution of the CMG helicase (Cdc45-MCM-GINS). Using Caenorhabditis elegans early embryos and Xenopus egg extracts, we show that the E3 ligase CUL-2LRR-1 associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 co-factors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2LRR1 as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2LRR-1, but is then removed by a mitotic pathway that requires the CDC-48 co-factor UBXN-3, orthologous to the human tumour suppressor FAF1. Partial inactivation of lrr-1 and ubxn-3 leads to synthetic lethality, suggesting future approaches by which a deeper understanding of CMG disassembly in metazoa could be exploited therapeutically.

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

“…Previous work showed that depletion of the orthologues of Ufd1 or Npl4 led to persistence of Cdc45 or GINS on mitotic chromatin in early embryos of the nematode Caenorhabditis elegans or in egg extracts of the frog Xenopus laevis (Franz et al., 2011). Although these findings were thought to reflect a post-replicative role for UFD1-NPL4-CDC48 in extracting GINS and Cdc45 (but not Mcm2–7) from chromatin during mitosis, we found that depletion of UFD-1, NPL-4, or CDC-48 in C. elegans early embryos leads to persistence of ubiquitylated CMG helicase (Sonneville et al., 2017). Moreover, UFD1-NPL4-p97 are recruited to chromatin during DNA replication termination in frog egg extracts (Dewar et al., 2017) and associate with ubiquitylated CMG helicase (Sonneville et al., 2017).…”

“…Although these findings were thought to reflect a post-replicative role for UFD1-NPL4-CDC48 in extracting GINS and Cdc45 (but not Mcm2–7) from chromatin during mitosis, we found that depletion of UFD-1, NPL-4, or CDC-48 in C. elegans early embryos leads to persistence of ubiquitylated CMG helicase (Sonneville et al., 2017). Moreover, UFD1-NPL4-p97 are recruited to chromatin during DNA replication termination in frog egg extracts (Dewar et al., 2017) and associate with ubiquitylated CMG helicase (Sonneville et al., 2017). These data indicate a conserved mechanism for CMG disassembly from yeast to animals.…”

SMC5/6 acts jointly with Fanconi anemia factors to support DNA repair and genome stability

“…The role of SMC5/6 in facilitating DNA repair may be manifested via recruitment and/or Sumoylation of BLM, already reported to act jointly with FANCD2 and SMC5/6/NSMCE2 in certain conditions of replication stress [21,64]. This latter envisaged function may involve the ubiquitin and SUMO ligase activity of the SMC5/6 complex and potentially facilitate replisome disassembly during replication termination [65] or cohesin removal to facilitate repair completion and normal chromosome structure in prophase [66]. This latter envisaged function may involve the ubiquitin and SUMO ligase activity of the SMC5/6 complex and potentially facilitate replisome disassembly during replication termination [65] or cohesin removal to facilitate repair completion and normal chromosome structure in prophase [66].…”

Human CST suppresses origin licensing and promotes AND-1/Ctf4 chromatin association

“…MCM is then unloaded from the chromatin following DNA synthesis in a ubiquitin-dependent manner (Dewar, Budzowska et al, 2015, Dewar, Low et al, 2017, Moreno, Bailey et al, 2014, Sonneville, Moreno et al, 2017. In initial experiments, we determined the effect of STN1 knockdown on the levels of chromatin-bound MCM in EdU positive versus negative cells ( Figure 1B and 2A).…”