Mitotic replisome disassembly depends on TRAIP ubiquitin ligase activity

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.

Download Full-text

Mitotic replisome disassembly in vertebrates

10.1101/418368 ◽

2018 ◽

Author(s):

Sara Priego Moreno ◽

Rebecca M. Jones ◽

Divyasree Poovathumkadavil ◽

Agnieszka Gambus

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Ubiquitin Ligase ◽

S Phase ◽

Replication Forks ◽

Replication Machinery ◽

Replicative Helicase ◽

Egg Extract ◽

Eukaryotic Dna ◽

Higher Eukaryotes

ABSTRACTRecent years have brought a breakthrough in our understanding of the process of eukaryotic DNA replication termination. We have shown that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in S-phase of the cell cycle is driven through polyubiquitylation of one of the replicative helicase subunits Mcm7. Our previous work in C.elegans embryos suggested also an existence of a back-up pathway of replisome disassembly in mitosis. Here we show, that in Xenopus laevis egg extract, any replisome retained on chromatin after S-phase is indeed removed from chromatin in mitosis. This mitotic disassembly pathway depends on formation of K6 and K63 ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and activity of p97/VCP protein segregase. The mitotic replisome pathway is therefore conserved through evolution in higher eukaryotes. However, unlike in lower eukaryotes it does not require SUMO modifications. This process can also remove any helicases from chromatin, including “active” stalled ones, indicating a much wider application of this pathway than just a “back-up” for terminated helicases.

Download Full-text

The Caenorhabditis elegans Replication Licensing Factor CDT-1 Is Targeted for Degradation by the CUL-4/DDB-1 Complex

Molecular and Cellular Biology ◽

10.1128/mcb.00736-06 ◽

2006 ◽

Vol 27 (4) ◽

pp. 1394-1406 ◽

Cited By ~ 35

Author(s):

Youngjo Kim ◽

Edward T. Kipreos

Keyword(s):

Caenorhabditis Elegans ◽

Ubiquitin Ligase ◽

S Phase ◽

Null Mutant ◽

Wild Type ◽

C Elegans ◽

Licensing Factor ◽

Ubiquitin Ligase E3 ◽

Blast Cells ◽

Temporal Restriction

ABSTRACT The replication of genomic DNA is strictly regulated to occur only once per cell cycle. This regulation centers on the temporal restriction of replication licensing factor activity. Two distinct ubiquitin ligase (E3) complexes, CUL4/DDB1 and SCFSkp2, have been reported to target the replication licensing factor Cdt1 for ubiquitin-mediated proteolysis. However, it is unclear to what extent these two distinct Cdt1 degradation pathways are conserved. Here, we show that Caenorhabditis elegans DDB-1 is required for the degradation of CDT-1 during S phase. DDB-1 interacts specifically with CUL-4 but not with other C. elegans cullins. A ddb-1 null mutant exhibits extensive DNA rereplication in postembryonic BLAST cells, similar to what is observed in cul-4(RNAi) larvae. DDB-1 physically associates with CDT-1, suggesting that CDT-1 is a direct substrate of the CUL-4/DDB-1 E3 complex. In contrast, a deletion mutant of the C. elegans Skp2 ortholog, skpt-1, appears overtly wild type with the exception of an impenetrant gonad migration defect. There is no appreciable role for SKPT-1 in the degradation of CDT-1 during S phase, even in a sensitized ddb-1 mutant background. We propose that the CUL-4/DDB-1 ubiquitin ligase is the principal E3 for regulating the extent of DNA replication in C. elegans.

Download Full-text

The intra-S phase checkpoint directly regulates replication elongation to preserve the integrity of stalled replisomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019183118 ◽

2021 ◽

Vol 118 (24) ◽

pp. e2019183118

Author(s):

Yang Liu ◽

Lu Wang ◽

Xin Xu ◽

Yue Yuan ◽

Bo Zhang ◽

...

Keyword(s):

Fission Yeast ◽

Dna Polymerases ◽

Replication Stress ◽

Underlying Mechanism ◽

S Phase ◽

Replication Forks ◽

Helicase Activity ◽

Replicative Helicase ◽

Checkpoint Kinases ◽

S Phase Checkpoint

DNA replication is dramatically slowed down under replication stress. The regulation of replication speed is a conserved response in eukaryotes and, in fission yeast, requires the checkpoint kinases Rad3ATR and Cds1Chk2. However, the underlying mechanism of this checkpoint regulation remains unresolved. Here, we report that the Rad3ATR-Cds1Chk2 checkpoint directly targets the Cdc45-MCM-GINS (CMG) replicative helicase under replication stress. When replication forks stall, the Cds1Chk2 kinase directly phosphorylates Cdc45 on the S275, S322, and S397 residues, which significantly reduces CMG helicase activity. Furthermore, in cds1Chk2-mutated cells, the CMG helicase and DNA polymerases are physically separated, potentially disrupting replisomes and collapsing replication forks. This study demonstrates that the intra-S phase checkpoint directly regulates replication elongation, reduces CMG helicase processivity, prevents CMG helicase delinking from DNA polymerases, and therefore helps preserve the integrity of stalled replisomes and replication forks.

Download Full-text

LRR1-mediated replisome disassembly promotes DNA replication by recycling replisome components

The Journal of Cell Biology ◽

10.1083/jcb.202009147 ◽

2021 ◽

Vol 220 (8) ◽

Author(s):

Yilin Fan ◽

Marielle S. Köberlin ◽

Nalin Ratnayeke ◽

Chad Liu ◽

Madhura Deshpande ◽

...

Keyword(s):

Enzyme Activity ◽

Cell Division ◽

Dna Replication ◽

Ubiquitin Ligase ◽

Essential Gene ◽

E3 Ubiquitin Ligase ◽

S Phase ◽

Replication Forks ◽

G2 Phase ◽

Rate Limiting

After two converging DNA replication forks meet, active replisomes are disassembled and unloaded from chromatin. A key process in replisome disassembly is the unloading of CMG helicases (CDC45–MCM–GINS), which is initiated in Caenorhabditis elegans and Xenopus laevis by the E3 ubiquitin ligase CRL2LRR1. Here, we show that human cells lacking LRR1 fail to unload CMG helicases and accumulate increasing amounts of chromatin-bound replisome components as cells progress through S phase. Markedly, we demonstrate that the failure to disassemble replisomes reduces the rate of DNA replication increasingly throughout S phase by sequestering rate-limiting replisome components on chromatin and blocking their recycling. Continued binding of CMG helicases to chromatin during G2 phase blocks mitosis by activating an ATR-mediated G2/M checkpoint. Finally, we provide evidence that LRR1 is an essential gene for human cell division, suggesting that CRL2LRR1 enzyme activity is required for the proliferation of cancer cells and is thus a potential target for cancer therapy.

Download Full-text

Replication-Coupled Chromatin Remodeling: An Overview of Disassembly and Assembly of Chromatin during Replication

International Journal of Molecular Sciences ◽

10.3390/ijms22031113 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1113

Author(s):

Céline Duc ◽

Christophe Thiriet

Keyword(s):

Cell Cycle ◽

Chromatin Remodeling ◽

Nuclear Import ◽

Genomic Dna ◽

Epigenetic Inheritance ◽

S Phase ◽

Chromatin Assembly ◽

Present Review ◽

Replication Forks ◽

Replication Machinery

The doubling of genomic DNA during the S-phase of the cell cycle involves the global remodeling of chromatin at replication forks. The present review focuses on the eviction of nucleosomes in front of the replication forks to facilitate the passage of replication machinery and the mechanism of replication-coupled chromatin assembly behind the replication forks. The recycling of parental histones as well as the nuclear import and the assembly of newly synthesized histones are also discussed with regard to the epigenetic inheritance.

Download Full-text

The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

10.1101/2021.12.16.472925 ◽

2021 ◽

Author(s):

Zeynep Tarcan ◽

Divyasree Poovathumkadavil ◽

Aggeliki Skagia ◽

Agnieszka Gambus

Keyword(s):

Dna Replication ◽

Molecular Mechanism ◽

Protein Complexes ◽

Ubiquitin Ligases ◽

S Phase ◽

E3 Ubiquitin Ligases ◽

Replication Machinery ◽

Replicative Helicase ◽

Cellular Processes ◽

Eukaryotic Dna

Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly in S-phase. Only Ubxn7 however facilitates efficient replisome disassembly through its interaction with both Cul2Lrr1 and p97. Our data therefore characterise Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates.

Download Full-text

Pivotal roles of PCNA loading and unloading on heterochromatin function

10.1101/232181 ◽

2017 ◽

Author(s):

Ryan Janke ◽

Grant King ◽

Martin Kupiec ◽

Jasper Rine

Keyword(s):

Dna Replication ◽

Transcriptional Silencing ◽

Structural Features ◽

S Phase ◽

Histone Chaperones ◽

Replication Forks ◽

Nucleosome Assembly ◽

Replication Machinery ◽

Loading And Unloading ◽

Regulatory Functions

ABSTRACTIn Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally-silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the PCNA unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.SIGNIFICANCE STATEMENTDNA replication poses a unique logistical challenge for the cell in that structural features of chromatin and their regulatory functions must be carefully coordinated with passage of replication machinery so faithful duplication of both the genome and its chromatin structures may be achieved. Nucleosome assembly is fundamental to reestablishment of chromatin in the wake of DNA replication, and here a mechanism by which nucleosome assembly is coordinated with DNA replication to maintain silenced chromatin is described.

Download Full-text

Cdt1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis

10.1101/2021.07.06.451311 ◽

2021 ◽

Author(s):

Nalin Ratnayeke ◽

Mingyu Chung ◽

Tobias Meyer

Keyword(s):

Dna Synthesis ◽

Ubiquitin Ligase ◽

Genome Instability ◽

S Phase ◽

Replication Forks ◽

Quantitative Microscopy ◽

Temporal Separation ◽

Origin Firing ◽

Origin Licensing ◽

Eukaryotic Dna

A fundamental concept in eukaryotic DNA replication is the temporal separation of G1 origin licensing from S phase origin firing. Re-replication and genome instability ensue if licensing occurs after DNA synthesis has started. In humans and other vertebrates, the E3 ubiquitin ligase CRL4Cdt2 starts to degrade the licensing factor Cdt1 after origins fire, raising the question of how cells prevent re-replication in early S phase. Here, using quantitative microscopy, we show that Cdt1 inhibits DNA synthesis during an overlap period when cells fire origins while Cdt1 is still present. Cdt1 inhibits DNA synthesis by suppressing CMG helicase progression at replication forks through the MCM-binding domain of Cdt1, and DNA synthesis commences once Cdt1 is degraded. Thus, instead of separating licensing from firing to prevent re-replication in early S phase, cells separate licensing from DNA synthesis through Cdt1-mediated inhibition of CMG helicase after firing.

Download Full-text