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The Journal of Biological Chemistry Nov 1984DNA polymerases induced by herpes simplex virus (HSV)-1 (KOS) and by three phosphonoformic acid-resistant strains were purified and the interaction of these enzymes with...
DNA polymerases induced by herpes simplex virus (HSV)-1 (KOS) and by three phosphonoformic acid-resistant strains were purified and the interaction of these enzymes with aphidicolin was examined. Incorporation of dATP, dCTP, and dTTP into activated DNA by parental enzyme was inhibited competitively by aphidicolin whereas dGTP incorporation was inhibited noncompetitively. Phosphonoformic acid-resistant enzymes were altered in KM and KI values for substrate and inhibitor, and two were inhibited by aphidicolin via the same modes as parental enzyme. However, aphidicolin competitively inhibited incorporation of dGTP by the third phosphonoformic acid-resistant enzyme under identical assay conditions. Two phosphonoformic acid-resistant enzymes were more sensitive than parental enzyme to inhibition by aphidicolin, indicating a close association between binding determinants for aphidicolin and for phosphonoformic acid on the virus DNA polymerase molecule. Aphidicolin inhibited hydrolysis of polynucleotide by HSV-1 DNA polymerase-associated nuclease. Inhibition was uncompetitive with DNA and the KI value (0.09 microM) was within the range of those calculated during nucleotide incorporation (0.071-0.74 microM). Therefore, aphidicolin may produce antiviral effects both by inhibition of deoxynucleotide incorporation and by deleterious effects resulting from inhibition of polymerase-associated nuclease.
Topics: Animals; Antiviral Agents; Aphidicolin; Cell Line; Chlorocebus aethiops; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Diterpenes; Exodeoxyribonucleases; Kidney; Kinetics; Nucleic Acid Synthesis Inhibitors; Protein Binding; Simplexvirus; Templates, Genetic; Viral Proteins
PubMed: 6092371
DOI: No ID Found -
Communications Biology Sep 2022Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT),...
Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT), causes replication arrest and cell death. Although CPT has excellent cytotoxicity, it has the disadvantage of instability under physiological conditions. Therefore, new types of TOP1 inhibitor have attracted particular attention. Here, we characterised the effect of a non-camptothecin inhibitor, Genz-644282 (Genz). First, we found that treatment with Genz showed cytotoxicity by introducing double-strand breaks (DSBs), which was suppressed by co-treatment with aphidicolin. Genz-induced DSB formation required the functions of TOP1. Next, we explored the advantages of Genz over CPT and found it was effective against CPT-resistant TOP1 carrying either N722S or N722A mutation. The effect of Genz was also confirmed at the cellular level using a CPT-resistant cell line carrying N722S mutation in the TOP1 gene. Moreover, we found arginine residue 364 plays a crucial role for the binding of Genz. Because tyrosine residue 723 is the active centre for DNA cleavage and re-ligation by TOP1, asparagine residue 722 plays crucial roles in the accessibility of the drug. Here, we discuss the mechanism of action of Genz on TOP1 inhibition.
Topics: Aphidicolin; Arginine; Asparagine; Camptothecin; DNA; DNA Topoisomerases, Type I; Naphthyridines; Tyrosine
PubMed: 36114357
DOI: 10.1038/s42003-022-03920-w -
Cell Cycle (Georgetown, Tex.) Oct 2019Chromosomal instability (CIN) causes structural and numerical chromosome aberrations and represents a hallmark of cancer. Replication stress (RS) has emerged as a driver...
Chromosomal instability (CIN) causes structural and numerical chromosome aberrations and represents a hallmark of cancer. Replication stress (RS) has emerged as a driver for structural chromosome aberrations while mitotic defects can cause whole chromosome missegregation and aneuploidy. Recently, first evidence indicated that RS can also influence chromosome segregation in cancer cells exhibiting CIN, but the underlying mechanisms remain unknown. Here, we show that chromosomally unstable cancer cells suffer from very mild RS, which allows efficient proliferation and which can be mimicked by treatment with very low concentrations of aphidicolin. Both, endogenous RS and aphidicolin-induced very mild RS cause chromosome missegregation during mitosis leading to the induction of aneuploidy. Moreover, RS triggers an increase in microtubule plus end growth rates in mitosis, an abnormality previously identified to cause chromosome missegregation in cancer cells. In fact, RS-induced chromosome missegregation is mediated by increased mitotic microtubule growth rates and is suppressed after restoration of proper microtubule growth rates and upon rescue of replication stress. Hence, very mild and cancer-relevant RS triggers aneuploidy by deregulating microtubule dynamics in mitosis.
Topics: Anaphase; Aneuploidy; Aphidicolin; Cell Line, Tumor; Cell Proliferation; Chromosomal Instability; Chromosome Segregation; DNA Damage; DNA Replication; Humans; Microtubules; Mitosis; Neoplasms
PubMed: 31448675
DOI: 10.1080/15384101.2019.1658477 -
Cell Reports Feb 20165-hydroxymethylcytosine (5hmC) is a DNA base created during active DNA demethylation by the recently discovered TET enzymes. 5hmC has essential roles in gene expression...
5-hydroxymethylcytosine (5hmC) is a DNA base created during active DNA demethylation by the recently discovered TET enzymes. 5hmC has essential roles in gene expression and differentiation. Here, we demonstrate that 5hmC also localizes to sites of DNA damage and repair. 5hmC accumulates at damage foci induced by aphidicolin and microirradiation and colocalizes with major DNA damage response proteins 53BP1 and γH2AX, revealing 5hmC as an epigenetic marker of DNA damage. Deficiency for the TET enzymes eliminates damage-induced 5hmC accumulation and elicits chromosome segregation defects in response to replication stress. Our results indicate that the TET enzymes and 5hmC play essential roles in ensuring genome integrity.
Topics: 5-Methylcytosine; Animals; Aphidicolin; CRISPR-Cas Systems; Cell Line; Cytosine; DNA Damage; DNA Methylation; DNA Repair; DNA Replication; DNA-Binding Proteins; Dioxygenases; Epigenesis, Genetic; Genome; Genomic Instability; HeLa Cells; Histones; Human Embryonic Stem Cells; Humans; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins; Rad51 Recombinase; Tumor Suppressor p53-Binding Protein 1
PubMed: 26854228
DOI: 10.1016/j.celrep.2016.01.035 -
The Journal of Cell Biology Aug 2021Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost...
Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress-response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle-TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle-TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.
Topics: Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Carrier Proteins; Cell Nucleolus; DNA Damage; DNA Replication; DNA, Ribosomal; DNA-Binding Proteins; Genomic Instability; HCT116 Cells; HeLa Cells; Humans; Hydroxyurea; Microscopy, Fluorescence; Nuclear Proteins; Phosphoproteins; Protein Binding; Protein Transport; Signal Transduction
PubMed: 34100862
DOI: 10.1083/jcb.202008085 -
DNA Repair Jun 2010Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the...
Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the DNA polymerase inhibitor, aphidicolin. We and others have identified several cell cycle checkpoint and DNA repair proteins that influence common fragile site stability. However, the initial events underlying fragile site breakage remain poorly understood. We demonstrate here that aphidicolin-induced gaps and breaks at fragile sites are prevented when cells are co-treated with low concentrations of the topoisomerase I inhibitor, camptothecin. This reduction in breakage is accompanied by a reduction in aphidicolin-induced RPA foci, CHK1 and RPA2 phosphorylation, and PCNA monoubiquitination, indicative of reduced levels of single stranded DNA. Furthermore, camptothecin reduces spontaneous fragile site breakage seen in cells lacking ATR, even in the absence of aphidicolin. These data from cultured human cells demonstrate that topoisomerase I activity is required for DNA common fragile site breaks and suggest that polymerase-helicase uncoupling is a key initial event in this process.
Topics: Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Camptothecin; Cell Cycle Proteins; Cell Nucleus; Checkpoint Kinase 1; Chromosome Breakage; Chromosome Fragile Sites; DNA Replication; DNA, Single-Stranded; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Pentacyclic Triterpenes; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Kinases; Protein Serine-Threonine Kinases; Replication Protein A; Topoisomerase I Inhibitors; Triterpenes; Ubiquitination; Betulinic Acid
PubMed: 20413351
DOI: 10.1016/j.dnarep.2010.03.005 -
Nature Communications Apr 2021The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around...
The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.
Topics: Aphidicolin; Calreticulin; Clonal Hematopoiesis; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Humans; Leukemia, Myeloid; Myeloid Progenitor Cells; Poly (ADP-Ribose) Polymerase-1; Repressor Proteins; Sequence Deletion; Serine-Arginine Splicing Factors; DNA Polymerase theta
PubMed: 33911081
DOI: 10.1038/s41467-021-22803-y -
FEBS Letters Mar 2021We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous...
We have previously demonstrated that Fanconi anemia (FA) proteins work in concert with other FA and non-FA proteins to mediate stalled replication fork restart. Previous studies suggest a connection between the FA protein FANCD2 and the non-FA protein mechanistic target of rapamycin (mTOR). A recent study showed that mTOR is involved in actin-dependent DNA replication fork restart, suggesting possible roles in the FA DNA repair pathway. In this study, we demonstrate that during replication stress mTOR interacts and cooperates with FANCD2 to provide cellular stability, mediate stalled replication fork restart, and prevent nucleolytic degradation of the nascent DNA strands. Taken together, this study unravels a novel functional cross-talk between two important mechanisms: mTOR and FA DNA repair pathways that ensure genomic stability.
Topics: Aphidicolin; Cell Survival; DNA; DNA Repair; DNA Replication; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Fibroblasts; Genome, Human; Genomic Instability; Humans; Hydroxyurea; Mitomycin; Primary Cell Culture; Protein Binding; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases
PubMed: 33423298
DOI: 10.1002/1873-3468.14035 -
Cancer Chemotherapy and Pharmacology Oct 2003Cytotoxicity from the anticancer drug 2',2'-difluoro-2'-deoxycytidine (dFdCyd) has been correlated with its incorporation into DNA. However, cytotoxicity may also result...
PURPOSE
Cytotoxicity from the anticancer drug 2',2'-difluoro-2'-deoxycytidine (dFdCyd) has been correlated with its incorporation into DNA. However, cytotoxicity may also result from inhibition of DNA synthesis, due to either (1) dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase, or (2) direct inhibition of DNA polymerases by the 5'-triphosphate of dFdCyd (dFdCTP). To elucidate the role of DNA synthesis inhibition in the cytotoxicity of dFdCyd, we compared dFdCyd to hydroxyurea (HU), a ribonucleotide reductase inhibitor, and aphidicolin, an inhibitor of DNA polymerases, in the U251 and D54 human glioblastoma cell lines.
METHODS
Sensitivity to dFdCyd, HU, and aphidicolin were determined using a colony formation assay. The effects of these drugs on DNA synthesis were measured by dual parameter flow cytometry, while the effects on nucleotide pool levels were analyzed by high-performance liquid chromatography.
RESULTS
HU and aphidicolin elicited substantially less cytotoxicity than the multi-log killing with dFdCyd. When used at equitoxic concentrations (24-h IC50 values), dFdCyd and HU decreased purine dNTP pools primarily, but dFdCyd was less effective than HU. dFdCyd had decreased dATP by about 80% after 4-12 h, and required 8-24 h to decrease DNA synthesis by 50%. In contrast, HU rapidly depleted dATP by >98% within 2 h, which resulted in >90% inhibition of DNA synthesis. Aphidicolin at a concentration similar to its Ki values for DNA polymerases (1 microM) decreased DNA synthesis by >70% within 2 h. However, this decreased cell survival by only 10% (U251 cells) and 40% (D54 cells).
CONCLUSIONS
These results demonstrate that HU and aphidicolin produced a more rapid and profound inhibition of DNA synthesis than dFdCyd, but resulted in significantly less cytotoxicity. This suggests that inhibition of DNA synthesis accounted for less than one log of the multi-log cytotoxicity observed with dFdCyd, whereas incorporation of dFdCTP into DNA is a more lethal event.
Topics: Antineoplastic Agents; Aphidicolin; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Chromatography, High Pressure Liquid; DNA, Neoplasm; Deoxycytidine; Enzyme Inhibitors; Glioblastoma; Humans; Hydroxyurea; Nucleic Acid Synthesis Inhibitors; Pyrimidine Nucleosides; Ribonucleotide Reductases; Gemcitabine
PubMed: 12811514
DOI: 10.1007/s00280-003-0661-5 -
American Journal of Respiratory Cell... Sep 2014We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to...
We recently proposed that mitotic asynchrony in repairing tissue may underlie chronic inflammation and fibrosis, where immune cell infiltration is secondary to proinflammatory cross-talk among asynchronously repairing adjacent tissues. Building on our previous finding that mitotic asynchrony is associated with proinflammatory/fibrotic cytokine secretion (e.g., transforming growth factor [TGF]-β1), here we provide evidence supporting cause-and-effect. Under normal conditions, primary airway epithelial basal cell populations undergo mitosis synchronously and do not secrete proinflammatory or profibrotic cytokines. However, when pairs of nonasthmatic cultures were mitotically synchronized at 12 hours off-set and then combined, the mixed cell populations secreted elevated levels of TGF-β1. This shows that mitotic asynchrony is not only associated with but is also causative of TGF-β1 secretion. The secreted cytokines and other mediators from asthmatic cells were not the cause of asynchronous regeneration; synchronously mitotic nonasthmatic epithelia exposed to conditioned media from asthmatic cells did not show changes in mitotic synchrony. We also tested if resynchronization of regenerating asthmatic airway epithelia reduces TGF-β1 secretion and found that pulse-dosed dexamethasone, simvastatin, and aphidicolin were all effective. We therefore propose a new model for chronic inflammatory and fibrotic conditions where an underlying factor is mitotic asynchrony.
Topics: Aphidicolin; Asthma; Bronchi; Cells, Cultured; Culture Media, Conditioned; Dexamethasone; Epithelial Cells; Epithelium; Fibrosis; Humans; Inflammation; Mitosis; Respiratory Mucosa; Simvastatin; Time Factors; Transforming Growth Factor beta1
PubMed: 24669775
DOI: 10.1165/rcmb.2013-0396OC