Regulation of Unperturbed DNA Replication by Ubiquitylation

Posttranslational modification of proteins by means of attachment of a small globular protein ubiquitin (i.e., ubiquitylation) represents one of the most abundant and versatile mechanisms of protein regulation employed by eukaryotic cells. Ubiquitylation influences almost every cellular process and its key role in coordination of the DNA damage response is well established. In this review we focus, however, on the ways ubiquitylation controls the process of unperturbed DNA replication. We summarise the accumulated knowledge showing the leading role of ubiquitin driven protein degradation in setting up conditions favourable for replication origin licensing and S-phase entry. Importantly, we also present the emerging major role of ubiquitylation in coordination of the active DNA replication process: preventing re-replication, regulating the progression of DNA replication forks, chromatin re-establishment and disassembly of the replisome at the termination of replication forks

A Decade of Discovery—Eukaryotic Replisome Disassembly at Replication Termination

Simple Summary: During cell division, DNA is duplicated through a process called DNA replication, so that each new cell inherits a copy of its own. A high level of accuracy is essential in this for the maintenance of genome stability and the prevention of genetic disorders and ageing-related diseases. In this review, we describe the current knowledge around DNA replication termination, in particular comparing and contrasting the proteins and mechanisms identified in different organisms—from archaea through to humans—but with a specific focus upon eukaryotic replication termination. We discuss when and where termination takes place, the mechanisms of replication fork convergence and the process of replisome disassembly, in both S-phase and in mitosis. Recent advances in the field have revealed high levels of regulation in the process of replisome disassembly, demonstrating the importance of timely and appropriate unloading of replication machinery. Finally, we summarise how replication termination defects may impact cellular health and raise questions to be addressed in the future within the field. Abstract: The eukaryotic replicative helicase (CMG complex) is assembled during DNA replication initiation in a highly regulated manner, which is described in depth by other manuscripts in this Issue. During DNA replication, the replicative helicase moves through the chromatin, unwinding DNA and facilitating nascent DNA synthesis by polymerases. Once the duplication of a replicon is complete, the CMG helicase and the remaining components of the replisome need to be removed from the chromatin. Research carried out over the last ten years has produced a breakthrough in our understanding, revealing that replication termination, and more specifically replisome disassembly, is indeed a highly regulated process. This review brings together our current understanding of these processes and highlights elements of the mechanism that are conserved or have undergone divergence throughout evolution. Finally, we discuss events beyond the classic termination of DNA replication in S-phase and go over the known mechanisms of replicative helicase removal from chromatin in these particular situations

Xenopus Mcm10 is a CDK-substrate required for replication fork stability

<p>During S phase, following activation of the S phase CDKs and the DBF4-dependent kinases (DDK), double hexamers of Mcm2-7 at licensed replication origins are activated to form the core replicative helicase. Mcm10 is one of several proteins that have been implicated from work in yeasts to play a role in forming a mature replisome during the initiation process. Mcm10 has also been proposed to play a role in promoting replisome stability after initiation has taken place. The role of Mcm10 is particularly unclear in metazoans, where conflicting data has been presented. Here, we investigate the role and regulation of Mcm10 in <i>Xenopus</i> egg extracts. We show that <i>Xenopus</i> Mcm10 is recruited to chromatin late in the process of replication initiation and this requires prior action of DDKs and CDKs. We also provide evidence that Mcm10 is a CDK substrate but does not need to be phosphorylated in order to associate with chromatin. We show that in extracts depleted of more than 99% of Mcm10, the bulk of DNA replication still occurs, suggesting that Mcm10 is not required for the process of replication initiation. However, in extracts depleted of Mcm10, the replication fork elongation rate is reduced. Furthermore, the absence of Mcm10 or its phosphorylation by CDK results in instability of replisome proteins on DNA, which is particularly important under conditions of replication stress.</p

Characterizing replisome disassembly in human cells

To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for “backup” mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes

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

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 laevis egg extracts, we show that the E3 ligase CUL-2(LRR-1) associates with the replisome and drives ubiquitylation and disassembly of CMG, together with the CDC-48 cofactors UFD-1 and NPL-4. Removal of CMG from chromatin in frog egg extracts requires CUL2 neddylation, and our data identify chromatin recruitment of CUL2(LRR1) as a key regulated step during DNA replication termination. Interestingly, however, CMG persists on chromatin until prophase in worms that lack CUL-2(LRR-1), but is then removed by a mitotic pathway that requires the CDC-48 cofactor 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