scholarly journals Mechanisms of eukaryotic replisome disassembly

2020 ◽  
Vol 48 (3) ◽  
pp. 823-836
Author(s):  
Sara Priego Moreno ◽  
Agnieszka Gambus
Keyword(s):  
Dna Replication ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Dna Lesions ◽  
Model Organisms ◽  
Replicative Helicase ◽  
Complex Process ◽  
Cross Links ◽  
Higher Eukaryotes ◽  
Key Events

DNA replication is a complex process that needs to be executed accurately before cell division in order to maintain genome integrity. DNA replication is divided into three main stages: initiation, elongation and termination. One of the key events during initiation is the assembly of the replicative helicase at origins of replication, and this mechanism has been very well described over the last decades. In the last six years however, researchers have also focused on deciphering the molecular mechanisms underlying the disassembly of the replicative helicase during termination. Similar to replisome assembly, the mechanism of replisome disassembly is strictly regulated and well conserved throughout evolution, although its complexity increases in higher eukaryotes. While budding yeast rely on just one pathway for replisome disassembly in S phase, higher eukaryotes evolved an additional mitotic pathway over and above the default S phase specific pathway. Moreover, replisome disassembly has been recently found to be a key event prior to the repair of certain DNA lesions, such as under-replicated DNA in mitosis and inter-strand cross-links (ICLs) in S phase. Although replisome disassembly in human cells has not been characterised yet, they possess all of the factors involved in these pathways in model organisms, and de-regulation of many of them are known to contribute to tumorigenesis and other pathological conditions.

scholarly journals Mitotic replisome disassembly in vertebrates

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.

scholarly journals Under-Replicated DNA: The Byproduct of Large Genomes?

Cancers ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 2764
Author(s):  
Agustina P. Bertolin ◽  
Jean-Sébastien Hoffmann ◽  
Vanesa Gottifredi
Keyword(s):  
Dna Replication ◽  
Genomic Dna ◽  
Genome Stability ◽  
S Phase ◽  
Nuclear Bodies ◽  
Eukaryotic Cells ◽  
End Joining ◽  
Higher Eukaryotes ◽  
Fork Stalling ◽  
Large Genomes

In this review, we provide an overview of how proliferating eukaryotic cells overcome one of the main threats to genome stability: incomplete genomic DNA replication during S phase. We discuss why it is currently accepted that double fork stalling (DFS) events are unavoidable events in higher eukaryotes with large genomes and which responses have evolved to cope with its main consequence: the presence of under-replicated DNA (UR-DNA) outside S phase. Particular emphasis is placed on the processes that constrain the detrimental effects of UR-DNA. We discuss how mitotic DNA synthesis (MiDAS), mitotic end joining events and 53BP1 nuclear bodies (53BP1-NBs) deal with such specific S phase DNA replication remnants during the subsequent phases of the cell cycle.

scholarly journals Transcription activity contributes to the firing of non-constitutive origins in African trypanosomes helping to maintain robustness in S-phase duration

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marcelo S. da Silva ◽  
Gustavo R. Cayres-Silva ◽  
Marcela O. Vitarelli ◽  
Paula A. Marin ◽  
Priscila M. Hiraiwa ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mechanistic Model ◽  
S Phase ◽  
Dna Lesions ◽  
Phase Duration ◽  
African Trypanosomes ◽  
Transcription Activity ◽  
Dna And Rna ◽  
Polycistronic Transcription ◽  
Dna Combing

AbstractThe co-synthesis of DNA and RNA potentially generates conflicts between replication and transcription, which can lead to genomic instability. In trypanosomatids, eukaryotic parasites that perform polycistronic transcription, this phenomenon and its consequences are still little studied. Here, we showed that the number of constitutive origins mapped in the Trypanosoma brucei genome is less than the minimum required to complete replication within S-phase duration. By the development of a mechanistic model of DNA replication considering replication-transcription conflicts and using immunofluorescence assays and DNA combing approaches, we demonstrated that the activation of non-constitutive (backup) origins are indispensable for replication to be completed within S-phase period. Together, our findings suggest that transcription activity during S phase generates R-loops, which contributes to the emergence of DNA lesions, leading to the firing of backup origins that help maintain robustness in S-phase duration. The usage of this increased pool of origins, contributing to the maintenance of DNA replication, seems to be of paramount importance for the survival of this parasite that affects million people around the world.

scholarly journals An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication

2019 ◽  
Vol 116 (27) ◽  
pp. 13374-13383 ◽  
Author(s):  
Tatiana N. Moiseeva ◽  
Yandong Yin ◽  
Michael J. Calderon ◽  
Chenao Qian ◽  
Sandra Schamus-Haynes ◽  
...  
Keyword(s):  
Dna Replication ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
S Phase ◽  
Dna Lesions ◽  
Dependent Manner ◽  
Kinase Signaling ◽  
Origin Firing ◽  
Atr Kinase ◽  
Chk1 Kinase

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

scholarly journals Mechanisms of genetic instability in a single S-phase following whole genome doubling

2021 ◽  
Author(s):  
Simon Gemble ◽  
Sara Vanessa Bernhard ◽  
Nishit Srivastava ◽  
Rene Wardenaar ◽  
Maddalena Nano ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genetic Instability ◽  
Chromosome Instability ◽  
Rapid Evolution ◽  
Cell Types ◽  
S Phase ◽  
Model Organisms ◽  
Whole Genome ◽  
Mass Scaling ◽  
Protein Mass

Doubling of the full chromosome content -whole genome duplications (WGDs)- is frequently found in human cancers and is responsible for the rapid evolution of genetically unstable karyotypes. It has previously been established that WGDs fuel chromosome instability due to abnormal mitosis owing to the presence of extra centrosomes and extra chromosomes. Tolerance to ploidy changes has been identified in different model organisms and cell types, revealing long term cellular adaptations that accommodate ploidy increase. Importantly, however, the immediate consequences of WGDs as cells become tetraploid are not known. It also remains unknown whether WGD triggers other events leading to genetic instability (GIN), independently of mitosis. In this study, we induced tetraploidy in diploid genetically stable RPE-1 cells and monitored the first interphase. We found that newly born tetraploids undergo high rates of DNA damage during DNA replication. Using DNA combing and single cell sequencing, we show that replication forks are unstable, perturbing DNA replication dynamics and generating under- and over-replicated regions at the end of S-phase. Mechanistically, we found that these defects result from lack of protein mass scaling up at the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can accumulate highly abnormal karyotypes. These findings provide an explanation for the GIN landscape that favors tumorigenesis after tetraploidization.

Are multiple checkpoint mediators involved in a checkpoint linking histone gene expression with DNA replication?

2007 ◽  
Vol 35 (5) ◽  
pp. 1369-1371 ◽  
Author(s):  
B. Müller ◽  
J. Blackburn ◽  
C. Feijoo ◽  
X. Zhao ◽  
C. Smythe
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Histone Gene ◽  
Control Mechanisms ◽  
Histone Genes ◽  
Checkpoint Kinases ◽  
Histone Gene Expression

In metazoans, accurate replication of chromosomes is ensured by the coupling of DNA synthesis to the synthesis of histone proteins. Expression of replication-dependent histone genes is restricted to S-phase by a combination of cell cycle-regulated transcriptional and post-transcriptional control mechanisms and is linked to DNA replication by a poorly understood mechanism involving checkpoint kinases [Su, Gao, Schneider, Helt, Weiss, O'Reilly, Bohmann and Zhao (2004) EMBO J. 23, 1133–1143; Kaygun and Marzluff (2005) Nat. Struct. Mol. Biol. 12, 794–800]. Here we propose a model for the molecular mechanisms that link these two important processes within S-phase, and propose roles for multiple checkpoints in this mechanism.

scholarly journals Replication fork dynamics and the DNA damage response

2012 ◽  
Vol 443 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Rebecca M. Jones ◽  
Eva Petermann
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Replication Fork ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Replication Initiation ◽  
Developmental Defects ◽  
Replication Fork Progression ◽  
Damage Response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

scholarly journals The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

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.

scholarly journals Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

2018 ◽  
Author(s):  
Alan Gao ◽  
Nicolai B. Larsen ◽  
Justin L. Sparks ◽  
Irene Gallina ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Dna Lesions ◽  
List Type ◽  
Genomic Integrity ◽  
Dna Metabolism ◽  
Single Stranded Dna ◽  
Egg Extracts ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

SummaryDNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.HighlightsThe proteasome, in addition to SPRTN, degrades DPCs during DNA replicationProteasome-dependent DPC degradation requires DPC ubiquitylationDPC ubiquitylation is triggered by ssDNA and does not require the replisomeSPRTN-dependent DPC degradation is a post-replicative process

scholarly journals Etoposide Induces the Dispersal of DNA Ligase I from Replication Factories

2001 ◽  
Vol 12 (7) ◽  
pp. 2109-2118 ◽  
Author(s):  
Alessandra Montecucco ◽  
Rossella Rossi ◽  
Giovanni Ferrari ◽  
A. Ivana Scovassi ◽  
Ennio Prosperi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ataxia Telangiectasia ◽  
Topoisomerase Ii ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Protein A ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Ligase ◽  
Dna Ligase I

In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA–cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.

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