Characterising replisome disassembly in human cells

Abstract

To ensure faultless duplication of the entire genome, eukaryotic replication initiates from thousands of replication origins. Replication forks emanating from origins move through the chromatin until they encounter forks from neighbouring origins, at which point they terminate. In the final stages of this process the replication machinery (replisome) is unloaded from chromatin and disassembled. Work from model organisms has elucidated that during replisome unloading, the MCM7 subunit of the terminated replicative helicase is polyubiquitylated and processed by p97/VCP segregase, leading to disassembly of the helicase and the replisome, which is built around it. In higher eukaryotes (worms, frogs, mouse embryonic stem cells), MCM7 ubiquitylation is driven by a Cullin2-based ubiquitin ligase, with LRR1 as a substrate receptor. To date, most of our knowledge of replication termination comes from model organisms and embryonic systems and little is known about how this process is executed and regulated in human somatic cells. Here we thus established methods to study replisome disassembly in human model cell lines. Using flow cytometry, immunofluorescence microscopy and chromatin isolation with western blotting, we can visualise unloading of the replisome (MCM7 and CDC45) from chromatin by the end of S-phase. We observe interaction of replicative helicase (CMG complex) with CUL2LRR1 and ubiquitylation of MCM7 on chromatin, specifically in S-phase, suggesting that this is a replication-dependent modification. Importantly, we are able to show that replisome disassembly in this system also requires Cullin2, LRR1 and p97, demonstrating conservation of the mechanism. Moreover, we present evidence that the back-up mitotic replisome disassembly pathway is also recapitulated in human somatic cells. Finally, while we find that treatment with small molecule inhibitors against cullin-based ubiquitin ligases (CULi) and p97 (p97i) does lead to phenotypes of replisome disassembly defects, they also both lead to induction of replication stress in somatic cells, which limits their usefulness as tools to specifically target replisome disassembly processes in this setting.

A Decade of Discovery—Eukaryotic Replisome Disassembly at Replication Termination

“…A similar back-up mitotic replisome disassembly pathway has now also been shown to exist in Xenopus laevis, mouse ES cells and human cells [66,68,98]. This pathway is capable of unloading replisomes at terminated or stalled replication forks and also relies on the same mechanisms/factors: ubiquitylation of MCM7 [98,99], CDC-48/p97/VCP unfoldase activity [64,68,98,99] and p97 cofactors NPL-4/UFD-1, as well as UBXN-3 in C. elegans [64], and UBXN7 and FAF1 in mouse ES cells [88] (Figure 5). Finally, while the evidence suggests that the SUMO protease ULP-4 supports this process in C. elegans [64], SUMOylation was found not to be required for the mitotic pathway functioning in X. laevis [98].…”

“…Experiments in C. elegans and X. laevis thus identified a Cullin ligase CUL-2 LRR−1 /Cul2 Lrr1 as a key protein recruited to chromatin at replication termination and confirmed that it was responsible for Mcm7 ubiquitylation [64,65]. More recent studies have now also confirmed CUL2 LRR1 to be needed for replisome disassembly in mouse stem cells and human cell lines [66][67][68] (Figure 3i). These latter studies demonstrated that the loss or depletion of LRR1 by CRISPR knockout, auxin-inducible degradation or siRNA impaired the ubiquitylation of MCM7 and led to the accumulation of replisomes on chromatin.…”

“…In support of this, the loss of LRR1 from human cells causes a reduction in the soluble levels of CDC45, POLE1 and TIMELESS and confers a significant reduction in global DNA synthesis [67]. Indeed, this latter phenotype has also been observed in cells lacking p97/CDC-48 activity [68,116] and it will be interesting to determine whether this reduction in DNA synthesis impacts late-firing replication origins in particular.…”

“…In contrast to yeast, C. elegans early embryos unload CMGs in prophase of mitosis, when the canonical S-phase pathway is perturbed [64]. A similar back-up mitotic replisome disassembly pathway has now also been shown to exist in Xenopus laevis, mouse ES cells and human cells [66,68,98]. This pathway is capable of unloading replisomes at terminated or stalled replication forks and also relies on the same mechanisms/factors: ubiquitylation of MCM7 [98,99], CDC-48/p97/VCP unfoldase activity [64,68,98,99] and p97 cofactors NPL-4/UFD-1, as well as UBXN-3 in C. elegans [64], and UBXN7 and FAF1 in mouse ES cells [88] (Figure 5).…”