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

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.

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

10.32920/ryerson.14664225.v1 ◽

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 ◽

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

The Journal of Cell Biology ◽

10.1083/jcb.200605080 ◽

2006 ◽

Vol 175 (5) ◽

pp. 729-741 ◽

Cited By ~ 61

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 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.

Identification and Characterization of Novel Small-Molecule Inhibitors of the Replication Checkpoint.

Blood ◽

10.1182/blood.v104.11.763.763 ◽

2004 ◽

Vol 104 (11) ◽

pp. 763-763

Author(s):

James Bradner ◽

Yong-Son Kim ◽

Angela Koehler ◽

Masaoki Kawasumi ◽

Xiaodong Li ◽

...

Keyword(s):

Cell Cycle ◽

Small Molecule ◽

Mammalian Cells ◽

Small Molecule Inhibitors ◽

Chromatin Condensation ◽

Replication Stress ◽

Chemotherapeutic Agents ◽

Replication Checkpoint ◽

Chromosomal Fragility ◽

Microarray Format

Abstract Background The replication (G2/M) checkpoint is principally mediated by the serine/threonine protein kinase ATR (ataxia telangiectasia mutated and Rad3-related). ATR is a large (350 kD) member of the phosphatidylinositol kinase related kinase family. After exposure to genotoxic or replication stress, ATR halts cell cycle progression, allowing DNA repair complexes time enough to restore the fidelity of the genome prior to cell division. Previous experiments have demonstrated that cancer cells with p53 mutation are critically dependent on ATR-mediated arrest of the cell cycle. Industrial approaches to identify ATR inhibitors have failed likely as a result of protein insolubility. Methods We have undertaken a novel chemical genetic approach employing small molecule microarrays (SMMs) to identify molecules with high binding specificity for ATR. Three diversity-oriented combinatorial chemical libraries of more than 15,000 entities were generated by split-pool synthesis in solid phase on polystyrene macrobead supports. Compounds were robotically printed in microarray format on glass slides. Four analogs of FK506 were printed as positive controls. Extracts were prepared from mammalian cells transfected with over-expression constructs of FLAG-tagged ATR, FKBP12 and GFP. A protocol was developed and optimized for screening employing a primary anti-FLAG mouse monoclonal antibody and Cy5-fluorophore labeled anti-mouse antibody. Data analysis for small molecule binders was performed with GenePix software on an Axon Scanner. Biological activity of these molecules was analyzed in the context of mitotic spread and chromosomal fragility assays. Results Protein expression and antibody fidelity was verified by Western blot. The lysate-based SMM screening approach was optimized and validated by recognition of an interaction between over-expressed, epitope-tagged FKBP12 and analogs of FK506. Six small molecule hits suggesting ATR binding were identified and verified by triplicate microarray assays. Positive compounds were structurally similar members of a dihydropyrancarboxamide library suggesting recognition of a common target. Mitotic spread analysis of cells treated with two of these molecules and hydroxyurea demonstrated the premature chromatin condensation phenotype characteristic of replication checkpoint inhibition. Chromosomal fragility was notably augmented by these molecules as well. Chemosensitivity following replication stress was witnessed in p53-negative cells relative to an otherwise identical wild-type cell line. Conclusions Classical approaches to drug discovery are often limited by challenges in protein biochemistry such as protein size, solubility, activity and yield. We present compelling data that the small molecule microarray format can effectively be tailored for use with cellular lysates over-expressing a protein target of biological interest. Furthermore, we have used an optimized protocol to identify two novel, active small molecule inhibitors of the replication checkpoint (SMIRC-1 and SMIRC-2). The enhanced chemosensitivity in p53-negative cell lines supports a plausible role for ATR inhibitors as potentially useful chemotherapeutic agents.

MDC1 collaborates with TopBP1 in DNA replication checkpoint control

The Journal of Cell Biology ◽

10.1083/jcb.201010026 ◽

2011 ◽

Vol 193 (2) ◽

pp. 267-273 ◽

Cited By ~ 66

Author(s):

Jiadong Wang ◽

Zihua Gong ◽

Junjie Chen

Keyword(s):

Dna Replication ◽

Cellular Response ◽

Checkpoint Control ◽

Dna Double Strand Breaks ◽

Replication Checkpoint ◽

Strand Breaks ◽

Major Player ◽

Cellular Dna ◽

Stalled Replication Forks

Human TopBP1 is a major player in the control of the DNA replication checkpoint. In this study, we identified MDC1, a key checkpoint protein involved in the cellular response to DNA double-strand breaks, as a TopBP1-associated protein. The specific TopBP1–MDC1 interaction is mediated by the fifth BRCT domain of TopBP1 and the Ser-Asp-Thr (SDT) repeats of MDC1. In addition, we demonstrated that TopBP1 accumulation at stalled replication forks is promoted by the H2AX/MDC1 signaling cascade. Moreover, MDC1 is important for ATR-dependent Chk1 activation in response to replication stress. Collectively, our data suggest that MDC1 facilitates several important steps in both cellular DNA damage response and the DNA replication checkpoint.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa963 ◽

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 ◽

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.

The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci

Genes & Development ◽

10.1101/gad.14.1.81 ◽

2000 ◽

Vol 14 (1) ◽

pp. 81-96 ◽

Cited By ~ 4

Author(s):

Christian Frei ◽

Susan M. Gasser

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Replication Fork ◽

Dna Helicase ◽

S Phase ◽

Replication Checkpoint ◽

Checkpoint Response ◽

Replication Fork Progression ◽

Cycle Arrest ◽

We have examined the cellular function of Sgs1p, a nonessential yeast DNA helicase, homologs of which are implicated in two highly debilitating hereditary human diseases (Werner's and Bloom's syndromes). We show that Sgs1p is an integral component of the S-phase checkpoint response in yeast, which arrests cells due to DNA damage or blocked fork progression during DNA replication. DNA polε and Sgs1p are found in the same epistasis group and act upstream of Rad53p to signal cell cycle arrest when DNA replication is perturbed. Sgs1p is tightly regulated through the cell cycle, accumulates in S phase and colocalizes with Rad53p in S-phase-specific foci, even in the absence of fork arrest. The association of Rad53p with a chromatin subfraction is Sgs1p dependent, suggesting an important role for the helicase in the signal-transducing pathway that monitors replication fork progression.

Managing Single-Stranded DNA during Replication Stress in Fission Yeast

10.32920/14637963 ◽

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.

The Spd1p S phase inhibitor can activate the DNA replication checkpoint pathway in fission yeast

Journal of Cell Science ◽

10.1242/jcs.113.23.4341 ◽

2000 ◽

Vol 113 (23) ◽

pp. 4341-4350 ◽

Cited By ~ 2

Author(s):

A. Borgne ◽

P. Nurse

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cell Cycle Progression ◽

S Phase ◽

Checkpoint Control ◽

Replication Checkpoint ◽

Checkpoint Pathway ◽

A Cell ◽

Checkpoint Genes

Spd1p (for S phase delayed) is a cell cycle inhibitor in Schizosaccharomyces pombe. Spd1p overexpression blocks the onset of both S phase and mitosis. In this study, we have investigated the mechanisms by which Spd1p overexpression blocks cell cycle progression, focussing on the block over mitotic onset. High levels of Spd1p lead to an increase in Y15 phosphorylation of Cdc2p and we show that the block over G(2) requires the Wee1p kinase and is dependent on the rad and chk1/cds1 checkpoint genes. We propose that high levels of Spd1p in G(2) cells activate the DNA replication checkpoint control, which leads to a Wee1p-dependent increase of Cdc2p Y15 phosphorylation blocking onset of mitosis. The Spd1p block at S phase onset may act by interfering directly with DNA replication, and also activates the G(2)rad/hus checkpoint pathway to block mitosis.

The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription

Molecular and Cellular Biology ◽

10.1128/mcb.00596-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 5977-5985 ◽

Cited By ~ 37

Author(s):

Chaitali Dutta ◽

Prasanta K. Patel ◽

Adam Rosebrock ◽

Anna Oliva ◽

Janet Leatherwood ◽

...

Keyword(s):

Transcription Factor ◽

Dna Replication ◽

Budding Yeast ◽

Replication Stress ◽

Phosphorylation Sites ◽

Transcriptional Program ◽

Replication Checkpoint ◽

Regulatory Similarity

ABSTRACT The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes.

Drosophila dCBP Is Involved in Establishing the DNA Replication Checkpoint

Molecular and Cellular Biology ◽

10.1128/mcb.01283-06 ◽

2006 ◽

Vol 27 (1) ◽

pp. 135-146 ◽

Cited By ~ 15

Author(s):

Sarah Smolik ◽

Kristen Jones

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Cell Cycle Arrest ◽

Chromatin Structure ◽

Genetic Interaction ◽

Structure And Function ◽

Replication Checkpoint ◽

Cycle Arrest ◽

And Function

ABSTRACT The CBP/p300 family of proteins comprises related acetyltransferases that coactivate signal-responsive transcription. Recent evidence suggests that p300/CBP may also interact directly with complexes that mediate different aspects of DNA metabolism such as replication and repair. In this report, we show that loss of dCBP in Drosophila cells and eye discs results in a defect in the cell cycle arrest induced by stalled DNA replication. We show that dCBP and the checkpoint kinase Mei-41 can be found together in a complex and, furthermore, that dCBP has a genetic interaction with mei-41 in the response to stalled DNA replication. These observations suggest a broader role for the p300/CBP acetyltransferases in the modulation of chromatin structure and function during DNA metabolic events as well as for transcription.