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Nucleic Acids Research Sep 2018G-quadruplex DNA has been viewed as a prospective anti-cancer target owing to its potential biological relevance. Real-time monitoring of DNA G-quadruplex structures in...
G-quadruplex DNA has been viewed as a prospective anti-cancer target owing to its potential biological relevance. Real-time monitoring of DNA G-quadruplex structures in living cells can provide valuable insights into the relationship between G-quadruplex formation and its cellular consequences. However, the probes capable of detecting DNA G-quadruplexes in living cells are still very limited. Herein, we reported a new fluorescent probe, IMT, for real-time visualization of DNA G-quadruplex structures in living cells. Using IMT as a fluorescent indicator, the quantity changes of DNA G-quadruplex at different points in time during continuous cellular progression responding to Aphidicolin and Hydroxyurea treatment have been directly visualized. Our data demonstrate that IMT will be a valuable tool for exploring DNA G-quadruplexes in live cells. Further application of IMT in fluorescence imaging may reveal more information on the roles of DNA G-quadruplexes in biological systems.
Topics: Aphidicolin; Cell Line, Tumor; DNA; Fluorescent Dyes; G-Quadruplexes; HeLa Cells; Humans; Hydroxyurea; Microscopy, Fluorescence; Spectrometry, Fluorescence
PubMed: 30085206
DOI: 10.1093/nar/gky665 -
The Korean Journal of Parasitology Apr 2019To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and...
To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.
Topics: Antiprotozoal Agents; Aphidicolin; Cell Cycle Checkpoints; Cell Cycle Proteins; G2 Phase; Gene Expression Profiling; Giardia lamblia; Nocodazole; Sequence Analysis, RNA; Up-Regulation
PubMed: 31104412
DOI: 10.3347/kjp.2019.57.2.185 -
Experimental Cell Research Jul 1995We have studied the effect of the cell synchronization agents compactin, ciclopirox olamine, mimosine, aphidicolin, ALLN, and colcemid on several parameters of cell...
We have studied the effect of the cell synchronization agents compactin, ciclopirox olamine, mimosine, aphidicolin, ALLN, and colcemid on several parameters of cell cycle progression in mitotically synchronized HeLa S3 cells. Using cell size and cyclin A and B levels as markers of cytoplasmic progression and DNA content as a measure of nuclear cell cycle position, we have examined coordination of cytoplasmic and nuclear events during induction synchrony. Each synchronizing agent was unique in its effect on the coordination of the cytoplasmic and nuclear cycle. Mimosine, aphidicolin, ALLN, and colcemid disrupted cell cycle integration while compactin and ciclopirox olamine did not. Continued net cell growth during cell cycle arrest was the most dramatic in aphidicolin-treated cells, which averaged a 60% increase in size. Mimosine, ALLN, and colcemid produced an increase in cell size of approximately 25%, and ciclopyrox olamine and compactin exerted a negligible effect. Cyclin A and B were found at mitotic (high) or G1 (low) levels, or in combination of high and low concentrations not correlated with DNA content in drug-treated cells. For example, treatment with mimosine, which arrests cells in G1 with 2C DNA, resulted in cyclin A accumulating to mitotic levels, whereas cyclin B remained at a low concentration, the first time this phenomenon has been observed. These results demonstrate that populations of synchronized cells obtained by different drug treatments are blocked at biochemically distinct cell cycle points not apparent by cytometric measurement of DNA content. Our results provide conclusive evidence that induced synchrony methods differ with respect to their impact on cell cycle organization and from the pattern seen with nonperturbing cell selection methods.
Topics: Aphidicolin; Cell Cycle; Cell Nucleus; Ciclopirox; Cyclins; Cytoplasm; DNA, Neoplasm; Demecolcine; HeLa Cells; Humans; Kinetics; Leupeptins; Mimosine; Protease Inhibitors; Pyridones; Time Factors
PubMed: 7628532
DOI: 10.1006/excr.1995.1216 -
Nucleic Acids Research Dec 2000Common fragile sites are chromosomal loci prone to breakage and rearrangement that can be induced by aphidicolin, an inhibitor of DNA polymerases. Within these loci,...
Common fragile sites are chromosomal loci prone to breakage and rearrangement that can be induced by aphidicolin, an inhibitor of DNA polymerases. Within these loci, sites of preferential DNA breaks were proposed to correlate with peaks of enhanced DNA flexibility, the function of which remains elusive. Here we show that mammalian DNA replication origins are enriched in peaks of enhanced flexibility. This finding suggests that the search for these features may help in the mapping of replication origins, and we present evidence supporting this hypothesis. The association of peaks of flexibility with replication origins also suggests that some origins may associate with minor levels of fragility. As shown here, an increased sensitivity to aphidicolin was found near two mammalian DNA replication origins.
Topics: Animals; Aphidicolin; Cell Line; Chromosome Breakage; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Mapping; Cricetinae; DNA; DNA Replication; In Situ Hybridization, Fluorescence; Replication Origin; Replicon; Tetrahydrofolate Dehydrogenase
PubMed: 11095694
DOI: 10.1093/nar/28.23.4805 -
Cell Structure and Function Jun 1998Methylation of cytosine in the genomic DNA plays an important role in mammalian embryogenesis. DNA methyltransferase activity, which contributes mainly to the...
Methylation of cytosine in the genomic DNA plays an important role in mammalian embryogenesis. DNA methyltransferase activity, which contributes mainly to the maintenance of the methylation pattern during proliferation, is under the control of the cell cycle, its activity being higher in the S phase than in the other phases (Adams, R.L.P., 1990, Biochem. J. 265, 309-320). In the present study, we examined how DNA methyltransferase is regulated in the cells arrested at S phase by aphidicolin treatment. The activity and protein levels of DNA methyltransferase in the nuclei were kept constant in proliferating mouse erythroleukemia cells, and increased about twofold after 6 h incubation in the presence of aphidicolin. This increase of DNA methyltransferase levels by aphidicolin treatment was positively correlated with the cell population at S phase. De novo synthesis of DNA methyltransferase protein was increased by the treatment. In addition, the relative half life of pulse labeled DNA methyltransferase was prolonged by aphidicolin treatment. Both increase in synthesis and prolongation of half life of DNA methyltransferase in S phase contributed to the increase of the activity and the protein levels by aphidicolin treatment. Prolongation of half life was abolished by cycloheximide, suggesting that newly synthesized protein(s) with a short half life participated in the degradation of DNA methyltransferase.
Topics: Animals; Aphidicolin; Cell Nucleus; DNA (Cytosine-5-)-Methyltransferases; Leukemia, Erythroblastic, Acute; Mice; S Phase; Tumor Cells, Cultured
PubMed: 9706402
DOI: 10.1247/csf.23.137 -
Genetics Jun 2021Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and...
Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
Topics: Animals; Aphidicolin; Cells, Cultured; DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Deoxyadenine Nucleotides; Dideoxynucleotides; Female; G2 Phase; Indoles; Mice; Nucleic Acid Synthesis Inhibitors; Oocytes; Primary Cell Culture; Rad51 Recombinase; Tetrahydroisoquinolines
PubMed: 33792683
DOI: 10.1093/genetics/iyab054 -
PloS One Apr 2011Micronuclei (MN) in mammalian cells serve as a reliable biomarker of genomic instability and genotoxic exposure. Elevation of MN is commonly observed in cells bearing...
BACKGROUND
Micronuclei (MN) in mammalian cells serve as a reliable biomarker of genomic instability and genotoxic exposure. Elevation of MN is commonly observed in cells bearing intrinsic genomic instability and in normal cells exposed to genotoxic agents. DNA double-strand breaks are marked by phosphorylation of H2AX at serine 139 (γ-H2AX). One subclass of MN contains massive and uniform γ-H2AX signals. This study tested whether this subclass of MN can be induced by replication stress.
PRINCIPAL FINDINGS
We observed that a large proportion of MN, from 20% to nearly 50%, showed uniform staining by antibodies against γ-H2AX, a marker of DNA double-strand breaks (DSBs). Such micronuclei were designated as MN-γ-H2AX (+). We showed that such MN can be induced by chemicals that are known to cause DNA replication stress and S phase arrest. Hydroxyurea, aphidicolin and thymidine could all significantly induce MN-γ-H2AX (+), which were formed during S phase and appeared to be derived from aggregation of DSBs. MN-γ-H2AX (-), MN that were devoid of uniform γ-H2AX signals, were induced to a lesser extent in terms of fold change. Paclitaxel, which inhibits the disassembly of microtubules, only induced MN-γ-H2AX (-). The frequency of MN-γ-H2AX (+), but not that of MN-γ-H2AX (-), was also significantly increased in cells that experience S phase prolongation due to depletion of cell cycle regulator CUL4B. Depletion of replication protein A1 (RPA1) by RNA interference resulted in an elevation of both MN-γ-H2AX (+) and MN-γ-H2AX (-).
CONCLUSIONS/SIGNIFICANCE
A subclass of MN, MN-γ-H2AX (+), can be preferentially induced by replication stress. Classification of MN according to their γ-H2AX status may provide a more refined evaluation of intrinsic genomic instabilities and the various environmental genotoxicants.
Topics: Animals; Aphidicolin; Cell Line; DNA Breaks, Double-Stranded; DNA Replication; Histones; Humans; Hydroxyurea; Mice; Micronuclei, Chromosome-Defective; Mutation; Paclitaxel; RNA, Small Interfering; Replication Protein A; S Phase; Stress, Physiological; Thymidine
PubMed: 21525980
DOI: 10.1371/journal.pone.0018618 -
Cell Feb 2016Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations...
Repair of DNA double-strand breaks (DSBs) by non-homologous end joining is critical for neural development, and brain cells frequently contain somatic genomic variations that might involve DSB intermediates. We now use an unbiased, high-throughput approach to identify genomic regions harboring recurrent DSBs in primary neural stem/progenitor cells (NSPCs). We identify 27 recurrent DSB clusters (RDCs), and remarkably, all occur within gene bodies. Most of these NSPC RDCs were detected only upon mild, aphidicolin-induced replication stress, providing a nucleotide-resolution view of replication-associated genomic fragile sites. The vast majority of RDCs occur in long, transcribed, and late-replicating genes. Moreover, almost 90% of identified RDC-containing genes are involved in synapse function and/or neural cell adhesion, with a substantial fraction also implicated in tumor suppression and/or mental disorders. Our characterization of NSPC RDCs reveals a basis of gene fragility and suggests potential impacts of DNA breaks on neurodevelopment and neural functions.
Topics: Animals; Aphidicolin; Basic Helix-Loop-Helix Transcription Factors; Brain; Cell Adhesion; Cell Adhesion Molecules, Neuronal; DNA Breaks; DNA End-Joining Repair; DNA Repair; GPI-Linked Proteins; Genome; Humans; Mice; Nerve Tissue Proteins; Neural Stem Cells; Synapses; Transcription Factors; Translocation, Genetic
PubMed: 26871630
DOI: 10.1016/j.cell.2015.12.039 -
Nucleic Acids Research Oct 2011Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A...
Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodelling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, γH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accumulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1-depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.
Topics: Aphidicolin; Apoptosis; Cell Line; Chromosomal Proteins, Non-Histone; Chromosome Fragility; DNA Damage; DNA Repair; G2 Phase Cell Cycle Checkpoints; Humans; M Phase Cell Cycle Checkpoints; RNA Interference; Transcription Factors
PubMed: 21745822
DOI: 10.1093/nar/gkr435 -
International Journal of Molecular... Feb 2022Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA...
Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.
Topics: Animals; Animals, Genetically Modified; Aphidicolin; CRISPR-Cas Systems; Cell Nucleus; DNA Replication; Embryo, Mammalian; Embryonic Development; Eukaryota; Gene Editing; Mosaicism; Swine; Zygote
PubMed: 35216252
DOI: 10.3390/ijms23042135