scholarly journals Cadmium (II)-Induced Oxidative Stress Results in Replication Stress and Epigenetic Modifications in Root Meristem Cell Nuclei of Vicia faba

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 640
Author(s):  
Aneta Żabka ◽  
Konrad Winnicki ◽  
Justyna Teresa Polit ◽  
Mateusz Wróblewski ◽  
Janusz Maszewski
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Dna ◽  
Chromatin Condensation ◽  
Replication Stress ◽  
Root Apical Meristem ◽  
H2o2 Production ◽  
Cell Nuclei ◽  
Meristem Cell

Among heavy metals, cadmium is considered one of the most toxic and dangerous environmental factors, contributing to stress by disturbing the delicate balance between production and scavenging of reactive oxygen species (ROS). To explore possible relationships and linkages between Cd(II)-induced oxidative stress and the consequent damage at the genomic level (followed by DNA replication stress), root apical meristem (RAM) cells in broad bean (V. faba) seedlings exposed to CdCl2 treatment and to post-cadmium recovery water incubations were tested with respect to H2O2 production, DNA double-strand breaks (γ-phosphorylation of H2AX histones), chromatin morphology, histone H3S10 phosphorylation on serine (a marker of chromatin condensation), mitotic activity, and EdU staining (to quantify cells typical of different stages of nuclear DNA replication). In order to evaluate Cd(II)-mediated epigenetic changes involved in transcription and in the assembly of nucleosomes during the S-phase of the cell cycle, the acetylation of histone H3 on lysine 5 (H3K56Ac) was investigated by immunofluorescence. Cellular responses to cadmium (II) toxicity seem to be composed of a series of interlinked biochemical reactions, which, via generation of ROS and DNA damage-induced replication stress, ultimately activate signal factors engaged in cell cycle control pathways, DNA repair systems, and epigenetic adaptations.

scholarly journals Day/Night Separation of Oxygenic Energy Metabolism and Nuclear DNA Replication in the Unicellular Red AlgaCyanidioschyzon merolae

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Shin-ya Miyagishima ◽  
Atsuko Era ◽  
Tomohisa Hasunuma ◽  
Mami Matsuda ◽  
Shunsuke Hirooka ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Dna Replication ◽  
Energy Metabolism ◽  
Cell Cycle Progression ◽  
Nuclear Dna ◽  
S Phase ◽  
Cycle Progression ◽  
Content Type

ABSTRACTThe transition from G1to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red algaCyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green algaChlamydomonas reinhardtii. Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation inC. merolae.IMPORTANCEEukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated inC. merolae. This sequestration enables “safe” proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.

scholarly journals Budding yeast Dma1 and Dma2 participate in regulation of Swe1 levels and localization

2011 ◽  
Vol 22 (13) ◽  
pp. 2185-2197 ◽  
Author(s):  
Erica Raspelli ◽  
Corinne Cassani ◽  
Giovanna Lucchini ◽  
Roberta Fraschini
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Budding Yeast ◽  
Cell Cycle Control ◽  
Replication Stress ◽  
Down Regulation ◽  
Evolutionarily Conserved ◽  
Dna Replication Stress ◽  
Morphogenesis Checkpoint ◽  
The One

Timely down-regulation of the evolutionarily conserved protein kinase Swe1 plays an important role in cell cycle control, as Swe1 can block nuclear division through inhibitory phosphorylation of the catalytic subunit of cyclin-dependent kinase. In particular, Swe1 degradation is important for budding yeast cell survival in case of DNA replication stress, whereas it is inhibited by the morphogenesis checkpoint in response to alterations in actin cytoskeleton or septin structure. We show that the lack of the Dma1 and Dma2 ubiquitin ligases, which moderately affects Swe1 localization and degradation during an unperturbed cell cycle with no apparent phenotypic effects, is toxic for cells that are partially defective in Swe1 down-regulation. Moreover, Swe1 is stabilized, restrained at the bud neck, and hyperphosphorylated in dma1Δ dma2Δ cells subjected to DNA replication stress, indicating that the mechanism stabilizing Swe1 under these conditions is different from the one triggered by the morphogenesis checkpoint. Finally, the Dma proteins are required for proper Swe1 ubiquitylation. Taken together, the data highlight a previously unknown role of these proteins in the complex regulation of Swe1 and suggest that they might contribute to control, directly or indirectly, Swe1 ubiquitylation.

The biphasic interphase-mitotic polarity of cell nuclei induced under DNA replication stress seems to be correlated with Pin2 localization in root meristems of Allium cepa

2015 ◽  
Vol 174 ◽  
pp. 62-70 ◽  
Author(s):  
Aneta Żabka ◽  
Paweł Trzaskoma ◽  
Konrad Winnicki ◽  
Justyna Teresa Polit ◽  
Agnieszka Chmielnicka ◽  
...  
Keyword(s):  
Dna Replication ◽  
Allium Cepa ◽  
Replication Stress ◽  
Cell Nuclei ◽  
Root Meristems ◽  
Dna Replication Stress

scholarly journals E2F1: Cause and Consequence of DNA Replication Stress

2021 ◽  
Vol 7 ◽  
Author(s):  
Shahd Fouad ◽  
David Hauton ◽  
Vincenzo D'Angiolella
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Tumor Suppressor ◽  
Signaling Pathways ◽  
Mammalian Cells ◽  
Replication Stress ◽  
Checkpoint Control ◽  
Oncogenic Signaling ◽  
Dna Replication Stress ◽  
Oncogenic Signaling Pathways

In mammalian cells, cell cycle entry occurs in response to the correct stimuli and is promoted by the transcriptional activity of E2F family members. E2F proteins regulate the transcription of S phase cyclins and genes required for DNA replication, DNA repair, and apoptosis. The activity of E2F1, the archetypal and most heavily studied E2F family member, is tightly controlled by the DNA damage checkpoints to modulate cell cycle progression and initiate programmed cell death, when required. Altered tumor suppressor and oncogenic signaling pathways often result in direct or indirect interference with E2F1 regulation to ensure higher rates of cell proliferation independently of external cues. Despite a clear link between dysregulated E2F1 activity and cancer progression, literature on the contribution of E2F1 to DNA replication stress phenotypes is somewhat scarce. This review discusses how dysfunctional tumor suppressor and oncogenic signaling pathways promote the disruption of E2F1 transcription and hence of its transcriptional targets, and how such events have the potential to drive DNA replication stress. In addition to the involvement of E2F1 upstream of DNA replication stress, this manuscript also considers the role of E2F1 as a downstream effector of the response to this type of cellular stress. Lastly, the review introduces some reflections on how E2F1 activity is integrated with checkpoint control through post-translational regulation, and proposes an exploitable tumor weakness based on this axis.

scholarly journals Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

2021 ◽  
Author(s):  
Cory Haluska ◽  
Fengzhi Jin ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Protein Phosphatase ◽  
Budding Yeast ◽  
Replication Stress ◽  
S Phase ◽  
Viability Loss ◽  
Phosphatase 2A ◽  
Dna Replication Stress

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK ( cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

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

Blood ◽  
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.

scholarly journals Nucleotide imbalance decouples cell growth from cell proliferation

2021 ◽  
Author(s):  
Frances F. Diehl ◽  
Teemu P. Miettinen ◽  
Ryan Elbashir ◽  
Christopher S. Nabel ◽  
Scott R. Manalis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Growth ◽  
Biomass Production ◽  
Cell Cycle Progression ◽  
Replication Stress ◽  
S Phase ◽  
Nucleotide Metabolism ◽  
Stress Signaling ◽  
Regulatory Pathways

AbstractNucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can accordingly inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is also not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways, causing aberrant biomass production and excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signaling to survive during, and recover from, nucleotide imbalance during S phase. In fact, replication stress signaling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signaling to cope with this metabolic problem, and disrupting the coordination of cell growth and division.

scholarly journals Centrosome impairment causes DNA replication stress through MLK3/MK2 signaling and R-loop formation

2020 ◽  
Author(s):  
Zainab Tayeh ◽  
Kim Stegmann ◽  
Antonia Kleeberg ◽  
Mascha Friedrich ◽  
Josephine Ann Mun Yee Choo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Stress ◽  
List Type ◽  
Replication Forks ◽  
Loop Formation ◽  
Animal Cells ◽  
Replication Fork Progression ◽  
Primordial Dwarfism ◽  
Dna Replication Stress

AbstractCentrosomes function as organizing centers of microtubules and support accurate mitosis in many animal cells. However, it remains to be explored whether and how centrosomes also facilitate the progression through different phases of the cell cycle. Here we show that impairing the composition of centrosomes, by depletion of centrosomal components or by inhibition of polo-like kinase 4 (PLK4), reduces the progression of DNA replication forks. This occurs even when the cell cycle is arrested before damaging the centrosomes, thus excluding mitotic failure as the source of replication stress. Mechanistically, the kinase MLK3 associates with centrosomes. When centrosomes are disintegrated, MLK3 activates the kinases p38 and MK2/MAPKAPK2. Transcription-dependent RNA:DNA hybrids (R-loops) are then causing DNA replication stress. Fibroblasts from patients with microcephalic primordial dwarfism (Seckel syndrome) harbouring defective centrosomes showed replication stress and diminished proliferation, which were each alleviated by inhibition of MK2. Thus, centrosomes not only facilitate mitosis, but their integrity is also supportive in DNA replication.HighlightsCentrosome defects cause replication stress independent of mitosis.MLK3, p38 and MK2 (alias MAPKAPK2) are signalling between centrosome defects and DNA replication stress through R-loop formation.Patient-derived cells with defective centrosomes display replication stress, whereas inhibition of MK2 restores their DNA replication fork progression and proliferation.Graphical abstract

scholarly journals DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

2012 ◽  
Vol 110 (8) ◽  
pp. 1581-1591 ◽  
Author(s):  
Aneta Żabka ◽  
Justyna Teresa Polit ◽  
Janusz Maszewski
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Allium Cepa ◽  
Replication Stress ◽  
Root Meristems ◽  
Dna Replication Stress
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