TY - UNPB
T1 - Characterising replisome disassembly in human cells
AU - Jones, R.M.
AU - Ruiz, J.H.
AU - Scaramuzza, S.
AU - Nath, S.
AU - Henklewska, M.
AU - Natsume, Toyoaki
AU - Romero, F.
AU - Kanemaki, M.T.
AU - Gambus, A.
PY - 2022/7/12
Y1 - 2022/7/12
N2 - 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.
AB - 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.
UR - https://www.biorxiv.org/content/10.1101/2022.07.12.499744v1
UR - http://www.scopus.com/inward/record.url?eid=2-s2.0-85136489396&partnerID=MN8TOARS
U2 - 10.1101/2022.07.12.499744
DO - 10.1101/2022.07.12.499744
M3 - Preprint
BT - Characterising replisome disassembly in human cells
ER -