scholarly journals Replication stress induced by CCNE1 overexpression creates a dependency on XRCC2 at the replication fork

2018 ◽  
Author(s):  
Kai Doberstein ◽  
Alison Karst ◽  
Paul T Kroeger ◽  
Ronny Drapkin
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
Strong Increase ◽  
Minichromosome Maintenance ◽  
Multiple Cancer ◽  
Synthetic Lethal ◽  
Cyclin E1 ◽  
Protein Levels ◽  
Cancer Types

SummaryAcross multiple cancer types, genome instability has been linked to aberrant over-expression of CCNE1 due to premature cell cycle entry and replication stress. Using a gain-of-function screen, we found that XRCC2 cooperates with CCNE1 in the neoplastic transformation of TP53 mutant cells. A pan-cancer analysis of TCGA data revealed a striking correlation between CCNE1 and XRCC2 expression and knockdown of XRCC2 in Cyclin E1 overexpressing cell lines is synthetic lethal. Immunopurification of XRCC2 showed that it interacts with the Minichromosome Maintenance Complex Component 7 (MCM7) protein. This interaction appears to be critical for protecting replication forks as knockdown of XRCC2 leads to a strong increase in MCM7 ubiquitination with concomitant decrease in MCM7 protein levels, and reduced replication fork speed. Importantly, Overexpression of MCM7 rescues the effect of XRCC2 knockdown. Our data describe a new dependency of Cyclin E1 overexpressing tumors on factors that stabilize the replication fork.

scholarly journals MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

2018 ◽  
Author(s):  
Emily Yun-chia Chang ◽  
James P. Wells ◽  
Shu-Huei Tsai ◽  
Yan Coulombe ◽  
Yujia A. Chan ◽  
...  
Keyword(s):  
Dna Replication ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Genome Instability ◽  
Human Cancer ◽  
Replication Stress ◽  
Maintenance Factors ◽  
Genome Wide ◽  
A Genome ◽  
Dna Replication Stress

SUMMARYEctopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

scholarly journals An essential role for dNTP homeostasis following CDK-induced replication stress

2018 ◽  
Author(s):  
Chen-Chun Pai ◽  
Kuo-Feng Hsu ◽  
Samuel. C. Durley ◽  
Andrea Keszthelyi ◽  
Stephen E. Kearsey ◽  
...  
Keyword(s):  
Ribonucleotide Reductase ◽  
Essential Role ◽  
Genome Instability ◽  
Supply And Demand ◽  
Replication Stress ◽  
Dna Integrity ◽  
Demand Model ◽  
Synthetic Lethal ◽  
Supply And Demand Model ◽  
Important Therapeutic Target

AbstractReplication stress is a common feature of cancer cells, and thus a potentially important therapeutic target. Here we show that CDK-induced replication stress is synthetic lethal with mutations disrupting dNTP homeostasis in fission yeast. Wee1 inactivation leads to increased dNTP demand and replication stress through CDK-induced firing of dormant replication origins. Subsequent dNTP depletion leads to inefficient DNA replication, Mus81-dependent DNA damage, and to genome instability. Cells respond to this replication stress by increasing dNTP supply through Set2-dependent MBF-induced expression of Cdc22, the catalytic subunit of ribonucleotide reductase (RNR). Disrupting dNTP synthesis following Wee1 inactivation, through abrogating Set2-dependent H3K36 tri-methylation or DNA integrity checkpoint inactivation results in critically low dNTP levels, replication collapse and cell death, which can be rescued by increasing dNTP levels. These findings support a ‘dNTP supply and demand’ model in which maintaining dNTP homeostasis is essential to prevent replication catastrophe in response to CDK-induced replication stress.

scholarly journals Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

2019 ◽  
Vol 218 (7) ◽  
pp. 2169-2184 ◽  
Author(s):  
Jacob Peter Matson ◽  
Amy M. House ◽  
Gavin D. Grant ◽  
Huaitong Wu ◽  
Joanna Perez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Live Cell Imaging ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycle Entry ◽  
Origin Licensing ◽  
Dna Replication Origins ◽  
Phase Entry

To maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is minichromosome maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading before S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. Consequently, the first S phase cells are hypersensitive to replication stress. This underlicensing results from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher-risk first cell cycle that likely promotes genome instability.

scholarly journals The dynamic equilibrium of nascent and parental MCMs safeguards replicating genomes

2019 ◽  
Author(s):  
Hana Sedlackova ◽  
Maj-Britt Rask ◽  
Rajat Gupta ◽  
Chunaram Choudhary ◽  
Kumar Somyajit ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Dynamic Equilibrium ◽  
Genome Instability ◽  
Replication Stress ◽  
Minichromosome Maintenance ◽  
Daughter Cells ◽  
Mild Reduction ◽  
Dividing Cells ◽  
High Level ◽  
Mother Cells

The MCM2-7 (minichromosome maintenance) protein complex is a DNA unwinding motor required for the eukaryotic genome duplication1. Although a huge excess of MCM2-7 is loaded onto chromatin in G1 phase to form pre-replication complexes (pre-RCs), only 5-10 percent are converted into a productive CDC45-MCM-GINS (CMG) helicase in S phase – a perplexing phenomenon often referred to as the ‘MCM paradox’2. Remaining pre-RCs stay dormant but can be activated under replication stress (RS)3. Remarkably, even a mild reduction in MCM pool results in genome instability4, 5, underscoring the critical requirement for high-level MCM maintenance to safeguard genome integrity across generations of dividing cells. How this is achieved remains unknown. Here, we show that for daughter cells to sustain error-free DNA replication, their mothers build up a stable nuclear pool of MCMs both by recycling of chromatin-bound MCMs (referred to as parental pool) and synthesizing new MCMs (referred to as nascent pool). We find that MCMBP, a distant MCM paralog6, ensures the influx of nascent MCMs to the declining recycled pool, and thereby sustains critical levels of MCMs. MCMBP promotes nuclear translocation of nascent MCM3-7 (but not MCM2), which averts accelerated MCM proteolysis in the cytoplasm, and thereby fosters assembly of licensing-competent nascent MCM2-7 units. Consequently, lack of MCMBP leads to reduction of nascent MCM3-7 subunits in mother cells, which translates to poor MCM inheritance and grossly reduced pre-RCs formation in daughter cells. Unexpectedly, whereas the pre-RC paucity caused by MCMBP deficiency does not alter the overall bulk DNA synthesis, it escalates the speed and asymmetry of individual replisomes. This in turn increases endogenous replication stress and renders cells hypersensitive to replication perturbations. Thus, we propose that surplus of MCMs is required to safeguard replicating genomes by modulating physiological dynamics of fork progression through chromatin marked by licensed but inactive MCM2-7 complexes.

scholarly journals Cdc48 targets INQ-localized Mrc1 to facilitate recovery from replication stress

2021 ◽  
Author(s):  
Camilla Colding ◽  
Jacob Autzen ◽  
Boris Pfander ◽  
Michael Lisby
Keyword(s):  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
Methyl Methanesulfonate ◽  
Replication Checkpoint ◽  
Replication Restart ◽  
Efficient Replication ◽  
Dna Replication Stress ◽  
Stress Mediator ◽  
Control Compartment

DNA replication stress is a source of genome instability and a replication checkpoint has evolved to enable fork stabilisation and completion of replication during stress. Mediator of the replication checkpoint 1 (Mrc1) is the primary mediator of this response in Saccharomyces cerevisiae. Mrc1 is partially sequestered in the intranuclear quality control compartment (INQ) upon methyl methanesulfonate (MMS)-induced replication stress. Here we show that Mrc1 re-localizes from the replication fork to INQ during replication stress. Sequestration of Mrc1 in INQ is facilitated by the Btn2 chaperone and the Cdc48 segregase is required to release Mrc1 from INQ during recovery from replication stress. Consistently, we show that Cdc48 colocalizes with Mrc1 in INQ and we find that Mrc1 is recognized by the Cdc48 cofactors Ufd1 and Otu1, which contribute to clearance of Mrc1 from INQ. Our findings suggest that INQ localization of Mrc1 and Cdc48 function to facilitate replication stress recovery by transiently sequestering the replication checkpoint mediator Mrc1 and explains our observation that Btn2 and Cdc48 are required for efficient replication restart following MMS-induced replication stress.

scholarly journals The oncoprotein DEK affects the outcome of PARP1/2 inhibition during replication stress

2019 ◽  
Author(s):  
Magdalena Ganz ◽  
Christopher Vogel ◽  
Christina Czada ◽  
Vera Jörke ◽  
Rebecca Kleiner ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Replication Fork ◽  
Replication Stress ◽  
Tumor Formation ◽  
Replication Forks ◽  
Functional Link ◽  
Protein Levels ◽  
Replication Fork Progression ◽  
Dna Replication Stress ◽  
Architectural Protein

ABSTRACTDNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

scholarly journals Managing Single-Stranded DNA during Replication Stress in Fission Yeast

2021 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Susan L. Forsburg
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Single Stranded Dna ◽  
Replication Checkpoint ◽  
Primary Signal ◽  
Fork Stalling ◽  
Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

scholarly journals Roles of Claspin in regulation of DNA replication, replication stress responses and oncogenesis in human cells

2021 ◽  
Author(s):  
Hao-Wen Hsiao ◽  
Chi-Chun Yang ◽  
Hisao Masai
Keyword(s):  
Dna Replication ◽  
Stress Responses ◽  
Replication Fork ◽  
Therapeutic Potential ◽  
Genome Instability ◽  
Intramolecular Interaction ◽  
Replication Stress ◽  
Human Cells ◽  
Entire Genome ◽  
Replication Checkpoint

AbstractHuman cells need to cope with the stalling of DNA replication to complete replication of the entire genome to minimize genome instability. They respond to “replication stress” by activating the conserved ATR-Claspin-Chk1 replication checkpoint pathway. The stalled replication fork is detected and stabilized by the checkpoint proteins to prevent disintegration of the replication fork, to remove the lesion or problems that are causing fork block, and to facilitate the continuation of fork progression. Claspin, a factor conserved from yeasts to human, plays a crucial role as a mediator that transmits the replication fork arrest signal from the sensor kinase, ataxia telangiectasia and Rad3-related (ATR), to the effector kinase, Checkpoint kinase 1 (Chk1). Claspin interacts with multiple kinases and replication factors and facilitates efficient replication fork progression and initiation during the normal course of DNA replication as well. It interacts with Cdc7 kinase through the acidic patch segment near the C-terminus and this interaction is critical for efficient phosphorylation of Mcm in non-cancer cells and also for checkpoint activation. Phosphorylation of Claspin by Cdc7, recruited to the acidic patch, regulates the conformation of Claspin through affecting the intramolecular interaction between the N- and C-terminal segments of Claspin. Abundance of Claspin is regulated at both mRNA and protein levels (post-transcriptional regulation and protein stability) and affects the extent of replication checkpoint. In this article, we will discuss how the ATR-Claspin-Chk1 regulates normal and stressed DNA replication and provide insight into the therapeutic potential of targeting replication checkpoint for efficient cancer cell death.

scholarly journals Managing Single-Stranded DNA during Replication Stress in Fission Yeast

2021 ◽  
Author(s):  
Sarah A. Sabatinos ◽  
Susan L. Forsburg
Keyword(s):  
Cellular Response ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Single Stranded Dna ◽  
Replication Checkpoint ◽  
Primary Signal ◽  
Fork Stalling ◽  
Chromosome Integrity

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

scholarly journals Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

2019 ◽  
Author(s):  
Jacob Peter Matson ◽  
Amy M. House ◽  
Gavin D. Grant ◽  
Huaitong Wu ◽  
Joanna Perez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Genome Instability ◽  
Replication Stress ◽  
S Phase ◽  
Minichromosome Maintenance ◽  
Cell Cycle Entry ◽  
Origin Licensing ◽  
Dna Replication Origin ◽  
Dna Replication Origins

SUMMARYThe authors find that human cells re-entering the cell cycle from quiescence have both an impaired p53-dependent DNA replication origin licensing checkpoint and slow origin licensing. This combination makes every first S phase underlicensed and hypersensitive to replication stress.ABSTRACTTo maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is Minichromosome Maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading prior to S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. As a result, the first S phase cells are hypersensitive to replication stress. This underlicensing is from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher risk cell cycle that promotes genome instability.

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