Abstract
We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2LRR1 ubiquitin ligase. Interestingly, upon inhibition of this pathway in Caenorhabditis elegans embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in Xenopus laevis egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a "backup" for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.
Highlights
Faithful cell division is the basis for the propagation of life and requires accurate duplication of all genetic information
We have shown that in Xenopus laevis egg extract and in Caenorhabditis elegans embryos, this replisome removal in S-phase is driven by Cul2LRR1 ubiquitin ligase, which ubiquitylates Mcm7 within the terminated CMG complex (Sonneville et al, 2017)
X. laevis egg extract is a cell-free system, which has proven to be instrumental over the years in studies of DNA replication
Summary
Faithful cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. The replication forks replicate chromatin until they encounter forks coming in opposite directions from neighbouring origins. We have shown that in Xenopus laevis egg extract and in Caenorhabditis elegans embryos, this replisome removal in S-phase is driven by Cul2LRR1 ubiquitin ligase, which ubiquitylates Mcm within the terminated CMG complex (Sonneville et al, 2017). Such modified CMG is recognised by p97/VCP segregase and removed from chromatin allowing for disassembly of the whole replisome built around the helicase (Moreno et al, 2014)
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