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 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 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 Bacillus subtilis RecA, DisA, and RadA/Sms Interplay Prevents Replication Stress by Regulating Fork Remodeling

2021 ◽  
Vol 12 ◽  
Author(s):  
Rubén Torres ◽  
Juan C. Alonso
Keyword(s):  
Bacillus Subtilis ◽  
Replication Fork ◽  
Replication Stress ◽  
Dna Helicase ◽  
Protein Protein Interaction ◽  
Branched Dna ◽  
Replication Restart ◽  
Negative Effect ◽  
Dna Replication Stress ◽  
Lagging Strand

Reviving Bacillus subtilis spores require the recombinase RecA, the DNA damage checkpoint sensor DisA, and the DNA helicase RadA/Sms to prevent a DNA replication stress. When a replication fork stalls at a template lesion, RecA filaments onto the lesion-containing gap and the fork is remodeled (fork reversal). RecA bound to single-strand DNA (ssDNA) interacts with and recruits DisA and RadA/Sms on the branched DNA intermediates (stalled or reversed forks), but DisA and RadA/Sms limit RecA activities and DisA suppresses its c-di-AMP synthesis. We show that RecA, acting as an accessory protein, activates RadA/Sms to unwind the nascent lagging-strand of the branched intermediates rather than to branch migrate them. DisA limits the ssDNA-dependent ATPase activity of RadA/Sms C13A, and inhibits the helicase activity of RadA/Sms by a protein-protein interaction. Finally, RadA/Sms inhibits DisA-mediated c-di-AMP synthesis and indirectly inhibits cell proliferation, but RecA counters this negative effect. We propose that the interactions among DisA, RecA and RadA/Sms, which are mutually exclusive, contribute to generate the substrate for replication restart, regulate the c-di-AMP pool and limit fork restoration in order to maintain cell survival.

scholarly journals Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae

2006 ◽  
Vol 175 (5) ◽  
pp. 729-741 ◽  
Author(s):  
Jorrit M. Enserink ◽  
Marcus B. Smolka ◽  
Huilin Zhou ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Cell Morphology ◽  
Replication Fork ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Proteins ◽  
Checkpoint Kinases ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Δ mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during 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 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 Translesion polymerase kappa-dependent DNA synthesis underlies replication fork recovery

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Peter Tonzi ◽  
Yandong Yin ◽  
Chelsea Wei Ting Lee ◽  
Eli Rothenberg ◽  
Tony T Huang
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Replication Fork ◽  
Genome Instability ◽  
Replication Stress ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Dna Replication Stress ◽  
Dependent Cell ◽  
Stalled Replication Forks

DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.

Helicases that interact with replication forks: new candidates from archaea

2005 ◽  
Vol 33 (6) ◽  
pp. 1471-1473 ◽  
Author(s):  
E.L. Bolt
Keyword(s):  
Cell Division ◽  
Dna Replication ◽  
Replication Fork ◽  
Genome Duplication ◽  
Genome Instability ◽  
Replication Forks ◽  
Valuable Resource ◽  
Replication Restart

Overcoming DNA replication fork blocks is essential for completing genome duplication and cell division. Archaea and eukaryotes drive replication using essentially the same protein machinery. Archaea may be a valuable resource for identifying new helicase components at advancing forks and/or in replication-restart pathways. As described here, these may be relevant to understanding genome instability in metazoans.

scholarly journals DNA replication stress induced by trifluridine determines tumor cell fate according to p53 status

2019 ◽  
Author(s):  
Yuki Kataoka ◽  
Makoto Iimori ◽  
Ryo Fujisawa ◽  
Tomomi Morikawa-Ichinose ◽  
Shinichiro Niimi ◽  
...  
Keyword(s):  
Dna Replication ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Fate ◽  
Replication Fork ◽  
Apoptotic Cell ◽  
Replication Stress ◽  
Dna Replication Stress ◽  
P53 Status ◽  
Fork Stalling

ABSTRACTDNA replication stress is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analog-type chemotherapeutic drugs introduce DNA damage and exacerbate DNA replication stress in tumor cells. However, the mechanisms underlying tumor cytotoxicity triggered by the drugs are not fully understood. Here, we show that the fluorinated thymidine analog trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases. FTD acted as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand. At the cellular level, FTD decreased thymidine triphosphate in the dNTP pool and induced FTD triphosphate accumulation, resulting in replication fork stalling caused by FTD incorporation into DNA. DNA lesions involving single-stranded DNA were generated as a result of replication fork stalling, and the p53-p21 pathway was activated. Although FTD suppressed tumor cell growth irrespective of p53 status, tumor cell fate diverged at the G2/M phase transition according to p53 status; tumor cells with wild-type p53 underwent cellular senescence via mitosis skip, whereas tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results suggest that DNA replication stress induced by a nucleoside analog-type chemotherapeutic drug triggers tumor cytotoxicity by determining tumor cell fate according to p53 status.SignificanceThis study identified a unique type of DNA replication stress induced by trifluridine, which directs tumor cell fate either toward cellular senescence or apoptotic cell death according to p53 status.

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