scholarly journals Active Replication Checkpoint Drives Genome Instability in Fission Yeast mcm4 Mutant

2020 ◽  
Vol 40 (14) ◽  
Author(s):  
Seong Min Kim ◽  
Susan L. Forsburg
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Genome Instability ◽  
Protein A ◽  
Replication Stress ◽  
Temperature Sensitive ◽  
Replication Checkpoint ◽  
Cycle Arrest ◽  
A Subunit ◽  
Replication Fork Arrest

ABSTRACT Upon replication fork arrest, the replication checkpoint kinase Cds1 is stimulated to preserve genome integrity. Robust activation of Cds1 in response to hydroxyurea prevents the endonuclease Mus81 from cleaving the stalled replication fork inappropriately. However, we find that the response is different in temperature-sensitive mcm4 mutants, affecting a subunit of the MCM replicative helicase. We show that Cds1 inhibition of Mus81 promotes genomic instability and allows mcm4-dg cells to evade cell cycle arrest. Cds1 regulation of Mus81 activity also contributes to the formation of the replication stress-induced DNA damage markers replication protein A (RPA) and Ku. These results identify a surprising role for Cds1 in driving DNA damage and disrupted chromosomal segregation under certain conditions of replication stress.

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 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 Chl1 helicase controls replication fork progression by regulating dNTP pools

2022 ◽  
Vol 5 (4) ◽  
pp. e202101153
Author(s):  
Amandine Batté ◽  
Sophie C van der Horst ◽  
Mireille Tittel-Elmer ◽  
Su Ming Sun ◽  
Sushma Sharma ◽  
...  
Keyword(s):  
Stress Response ◽  
Replication Fork ◽  
Replication Stress ◽  
Stress Conditions ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Replication Fork Progression ◽  
Intracellular Dntp ◽  
Replication Fork Arrest

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

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 PHF2 histone demethylase prevents DNA damage and genome instability by controlling cell cycle progression of neural progenitors

2019 ◽  
Vol 116 (39) ◽  
pp. 19464-19473 ◽  
Author(s):  
Stella Pappa ◽  
Natalia Padilla ◽  
Simona Iacobucci ◽  
Marta Vicioso ◽  
Elena Álvarez de la Campa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Genome Instability ◽  
Neural Progenitors ◽  
Cycle Progression ◽  
Cycle Arrest ◽  
Biochemical Assays

Histone H3 lysine 9 methylation (H3K9me) is essential for cellular homeostasis; however, its contribution to development is not well established. Here, we demonstrate that the H3K9me2 demethylase PHF2 is essential for neural progenitor proliferation in vitro and for early neurogenesis in the chicken spinal cord. Using genome-wide analyses and biochemical assays we show that PHF2 controls the expression of critical cell cycle progression genes, particularly those related to DNA replication, by keeping low levels of H3K9me3 at promoters. Accordingly, PHF2 depletion induces R-loop accumulation that leads to extensive DNA damage and cell cycle arrest. These data reveal a role of PHF2 as a guarantor of genome stability that allows proper expansion of neural progenitors during development.

scholarly journals Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

2013 ◽  
Vol 33 (16) ◽  
pp. 3390-3390
Author(s):  
Mayank Singh ◽  
Clayton R. Hunt ◽  
Raj K. Pandita ◽  
Rakesh Kumar ◽  
Chin-Rang Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Lamin A ◽  
Replication Fork Arrest

scholarly journals A Mutant Allele of MRE11 Found in Mismatch Repair-deficient Tumor Cells Suppresses the Cellular Response to DNA Replication Fork Stress in a Dominant Negative Manner

2008 ◽  
Vol 19 (4) ◽  
pp. 1693-1705 ◽  
Author(s):  
Qin Wen ◽  
Jennifer Scorah ◽  
Geraldine Phear ◽  
Gary Rodgers ◽  
Sheila Rodgers ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mutant Allele ◽  
Cellular Response ◽  
Replication Fork ◽  
Protein A ◽  
Cancer Cell Line ◽  
Replication Stress ◽  
Dominant Negative ◽  
Mutant Protein ◽  
In Cells

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3′-5′ exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3′ single-strand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time- and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.

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