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

Abstract

SummaryDisassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.

TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts

“…This is likely because p97 has pleiotropic roles in DNA replication and can be recruited to replication forks by different p97 cofactors to, for example, regulate DNA replication origin firing or promote DNA replication termination by unloading the CMG helicase ( Supplementary Fig. 7F) [67][68][69][70][71] .…”

USP7 couples DNA replication termination to mitotic entry

“…Moreover, MCM7 ubiquitination was also detected in control RPE cells at time-points coincident with mitotic entry and thus with the completion of DNA replication (8-12 hrs). Upon its ubiquitination at the end of DNA replication, MCM7 is extracted from chromatin by the action of the VCP segregase (Maric et al, 2017, Sonneville et al, 2017. Likewise, USP7 inhibition led to an accumulation of the VCP segregase on chromatin concomitant with MCM7 ubiquitination ( Figure 1B).…”

“…We now know that the dissociation of the CMG helicase (CDC45, MCM2-7 and GINS) is a key event in termination, which only occurs after the DNA from two converging forks is fully replicated and ligated (Dewar, Budzowska et al, 2015). At the molecular level, replisome disassembly requires MCM7 ubiquitination (Maric, Maculins et al, 2014, Moreno, Bailey et al, 2014, which drives its extraction from chromatin by the VCP segregase and its adaptors UFD1L and NPLOC4 (Maric, Mukherjee et al, 2017, Sonneville, Moreno et al, 2017. In addition, the unloading of the MCM complex in late S-phase is facilitated by MCM-BP (Nishiyama, Frappier et al, 2011).…”

“…Proteomic analyses revealed that USP7 inhibition increases the ubiquitination of SUMO and SUMOylated factors, which are displaced from replication factories. Interestingly, MCM7 was one of the proteins to show increased ubiquitination upon USP7 inhibition, with the modification occurring at the same region that had been linked to DNA replication termination in yeast (Maric et al, 2017). Moreover, prior work showed that MCM-BP tethers USP7 to MCM proteins, and that lack of USP7 increases the levels of chromatin-bound MCM (Jagannathan, Nguyen et al, 2014).…”

Cdc48 regulates intranuclear quality control sequestration of the Hsh155 splicing factor

“…S1D) (16,17). In addition, Cdc48 regulates protein-DNA complexes, such as the removal of RNA polymerase from damaged DNA (15), the removal of replisomes during DNA replication termination (14), at protein-DNA crosslinks (21), and in the release of condensin complexes (22). Thus, Cdc48 is well-positioned to regulate protein sequestration pathways such as INQ following DNA damage detection.…”