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 Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

2020 ◽  
Vol 48 (21) ◽  
pp. 12169-12187
Author(s):  
Rose Westhorpe ◽  
Andrea Keszthelyi ◽  
Nicola E Minchell ◽  
David Jones ◽  
Jonathan Baxter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Stress ◽  
Dna Helicase ◽  
The Other ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Binding Partner ◽  
Dna Replication Checkpoint

Abstract The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

scholarly journals Functions of Saccharomyces cerevisiae 14-3-3 Proteins in Response to DNA Damage and to DNA Replication Stress

Genetics ◽  
2003 ◽  
Vol 165 (4) ◽  
pp. 1717-1732
Author(s):  
Francisca Lottersberger ◽  
Fabio Rubert ◽  
Veronica Baldo ◽  
Giovanna Lucchini ◽  
Maria Pia Longhese
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Instability ◽  
Chromosomal Rearrangements ◽  
Replication Stress ◽  
Checkpoint Kinases ◽  
Gross Chromosomal Rearrangements ◽  
Dna Damage Checkpoints ◽  
Dna Replication Stress

Abstract Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Δ mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polα-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Δ and bmh1-170 bmh2Δ mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability.

A mechanism for Rad53 to couple leading- and lagging-strand DNA synthesis under replication stress in budding yeast

2021 ◽  
Vol 118 (38) ◽  
pp. e2109334118
Author(s):  
Albert Serra-Cardona ◽  
Chuanhe Yu ◽  
Xinmin Zhang ◽  
Xu Hua ◽  
Yuan Yao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Replication Stress ◽  
Replication Checkpoint ◽  
Checkpoint Pathway ◽  
Dna Replication Checkpoint ◽  
Dna Replication Stress ◽  
Leading Strand ◽  
Mutant Cells ◽  
Lagging Strand

In response to DNA replication stress, DNA replication checkpoint kinase Mec1 phosphorylates Mrc1, which in turn activates Rad53 to prevent the generation of deleterious single-stranded DNA, a process that remains poorly understood. We previously reported that lagging-strand DNA synthesis proceeds farther than leading strand in rad53-1 mutant cells defective in replication checkpoint under replication stress, resulting in the exposure of long stretches of the leading-strand templates. Here, we show that asymmetric DNA synthesis is also observed in mec1-100 and mrc1-AQ cells defective in replication checkpoint but, surprisingly, not in mrc1∆ cells in which both DNA replication and checkpoint functions of Mrc1 are missing. Furthermore, depletion of either Mrc1 or its partner, Tof1, suppresses the asymmetric DNA synthesis in rad53-1 mutant cells. Thus, the DNA replication checkpoint pathway couples leading- and lagging-strand DNA synthesis by attenuating the replication function of Mrc1-Tof1 under replication stress.

scholarly journals Separable functions of Tof1/Timeless in intra-S-checkpoint signalling, replisome stability and DNA topological stress

2019 ◽  
Author(s):  
Rose Westhorpe ◽  
Andrea Keszthelyi ◽  
Nicola E. Minchell ◽  
David Jones ◽  
Jonathan Baxter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Replication Stress ◽  
Dna Helicase ◽  
The Other ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Binding Partner ◽  
Dna Replication Checkpoint

AbstractThe highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.

scholarly journals Tolerance of DNA Replication Stress Is Promoted by Fumarate Through Modulation of Histone Demethylation and Enhancement of Replicative Intermediate Processing in Saccharomyces cerevisiae

Genetics ◽  
2019 ◽  
Vol 212 (3) ◽  
pp. 631-654 ◽  
Author(s):  
Faeze Saatchi ◽  
Ann L. Kirchmaier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Cell Cancer ◽  
Replication Stress ◽  
Histone Demethylase ◽  
Loss Of Function ◽  
Cycle Enzyme ◽  
Link Type ◽  
Dna Replication Stress

Fumarase is a well-characterized TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate. In mammals, fumarase acts as a tumor suppressor, and loss-of-function mutations in the FH gene in hereditary leiomyomatosis and renal cell cancer result in the accumulation of intracellular fumarate—an inhibitor of α-ketoglutarate-dependent dioxygenases. Fumarase promotes DNA repair by nonhomologous end joining in mammalian cells through interaction with the histone variant H2A.Z, and inhibition of KDM2B, a H3 K36-specific histone demethylase. Here, we report that Saccharomyces cerevisiae fumarase, Fum1p, acts as a response factor during DNA replication stress, and fumarate enhances survival of yeast lacking Htz1p (H2A.Z in mammals). We observed that exposure to DNA replication stress led to upregulation as well as nuclear enrichment of Fum1p, and raising levels of fumarate in cells via deletion of FUM1 or addition of exogenous fumarate suppressed the sensitivity to DNA replication stress of htz1Δ mutants. This suppression was independent of modulating nucleotide pool levels. Rather, our results are consistent with fumarate conferring resistance to DNA replication stress in htz1Δ mutants by inhibiting the H3 K4-specific histone demethylase Jhd2p, and increasing H3 K4 methylation. Although the timing of checkpoint activation and deactivation remained largely unaffected by fumarate, sensors and mediators of the DNA replication checkpoint were required for fumarate-dependent resistance to replication stress in the htz1Δ mutants. Together, our findings imply metabolic enzymes and metabolites aid in processing replicative intermediates by affecting chromatin modification states, thereby promoting genome integrity.

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.

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 Measuring the effect of environmental stress on cell survival during replication stress

2021 ◽  
Author(s):  
Poojaben Patel
Keyword(s):  
Cell Cycle ◽  
Heat Shock ◽  
Environmental Stress ◽  
Cellular Response ◽  
Replication Stress ◽  
Environmental Stressors ◽  
Replication Checkpoint ◽  
Checkpoint Proteins ◽  
Cellular Environment ◽  
Dna Replication Checkpoint

DNA replication checkpoint ensures cell fitness under replication stress by restraining fork progression and arresting cell cycle. Without checkpoint proteins, cells die in a replication inhibitor hydroxyurea (HU). However, cellular environment may affect their survival in HU. Therefore, the main goal of this study was to examine the effect of environmental stress and to study how it promotes survival in replication checkpoint mutants of fission yeast (rad3∆, mrc1∆, cds1∆). Our viability assays showed a significant increase in these mutants survival in heat-shock + HU compared to HU alone. Cell-cycle staging suggests that cells are altered after heat shock, affecting their response to HU. We measured the consequences to this enhanced survival and found that surviving population exhibits altered DNA mis-segregation and mutation rate. Collectively, our work points to a general cellular response to various environmental stressors that affects survival under replication stress, and may be applicable to human disease.

scholarly journals A FOXO-dependent replication checkpoint restricts proliferation of damaged cells

2020 ◽  
Author(s):  
Marten Hornsveld ◽  
Femke M Feringa ◽  
Lenno Krenning ◽  
Jeroen van den Berg ◽  
Lydia MM Smits ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Stress ◽  
Anaphase Promoting Complex ◽  
Endogenous Factors ◽  
Replication Checkpoint ◽  
Checkpoint Response ◽  
Exit Mechanism ◽  
Forkhead Box ◽  
M Phase ◽  
Dna Replication Stress

AbstractDNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Stalling or slowing of the replication fork as a result of replication stress leads to formation of aberrant single-stranded DNA (ssDNA) stretches and potentially DNA double-stranded-breaks (DSBs). Accumulation of ssDNA activates the ATR-dependent DNA replication stress checkpoint response that slows progression from S/G2- to M-phase to protect genomic integrity (1). However, whether mild replication stress restricts proliferation remains controversial (2–6). Here we identify a novel cell cycle exit mechanism, that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the CDH1 bound Anaphase Promoting Complex / Cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead Box O (FOXOs) mediated repression of its transcriptional regulator E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible, and thereby play an important role in maintaining genomic stability.

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