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Nucleic Acids Research May 2024In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much...
In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
Topics: Humans; Antigens, Neoplasm; Carbazoles; Chromatin; Diketopiperazines; DNA; DNA Damage; DNA Topoisomerases, Type II; DNA-Binding Proteins; High Mobility Group Proteins; Histones; Intercalating Agents; Poly-ADP-Ribose Binding Proteins; RNA Polymerase I; RNA Polymerase II; RNA Polymerase III; Topoisomerase II Inhibitors; Transcription, Genetic; Transcriptional Elongation Factors; Aclarubicin
PubMed: 38340348
DOI: 10.1093/nar/gkae069 -
Molecular Microbiology Jan 2023Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This... (Review)
Review
Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This pattern of diversity reflects, in part, the burst-like nature of transcription. Transcription bursting has many causes and a failure to remove the supercoils that accumulate in DNA during transcription elongation is an important contributor. Positive supercoiling of the DNA ahead of the transcription elongation complex can result in RNA polymerase stalling if this DNA topological roadblock is not removed. The relaxation of these positive supercoils is performed by the ATP-dependent type II topoisomerases DNA gyrase and topoisomerase IV. Interference with the action of these topoisomerases involving, inter alia, topoisomerase poisons, fluctuations in the [ATP]/[ADP] ratio, and/or the intervention of nucleoid-associated proteins with GapR-like or YejK-like activities, may have consequences for the smooth operation of the transcriptional machinery. Antibiotic-tolerant (but not resistant) persister cells are among the phenotypic outliers that may emerge. However, interference with type II topoisomerase activity can have much broader consequences, making it an important epigenetic driver of physiological diversity in the bacterial population.
Topics: DNA; DNA Gyrase; DNA Topoisomerase IV; Bacteria; DNA Topoisomerases, Type I; Adenosine Triphosphate; Epigenesis, Genetic; DNA, Superhelical; DNA, Bacterial
PubMed: 36565252
DOI: 10.1111/mmi.15014 -
Molecules (Basel, Switzerland) Nov 2022Novel semisynthetic coumarin derivatives were synthesized to be developed as chemotherapeutic anticancer agents through topoisomerase II, VEGFR2 inhibition that leads to...
Novel semisynthetic coumarin derivatives were synthesized to be developed as chemotherapeutic anticancer agents through topoisomerase II, VEGFR2 inhibition that leads to apoptotic cancer cell death. The coumarin amino acids and dipeptides derivatives were prepared by the reaction of coumarin-3-carboxylic acid with amino acid methyl esters following the ,-dicyclohexylcarbodiimide (DCC) method and 1-hydroxy-benzotriazole (HOBt), as coupling reagents. The synthesized compounds were screened towards VEGFR2, and topoisomerase IIα proteins to highlight their binding affinities and virtual mechanism of binding. Interestingly, compounds (Tyr) and (β-Ala-L-Met) shared the activity towards the three proteins by forming the same interactions with the key amino acids, such as the co-crystallized ligands. Both compounds and exhibited potent cytotoxic activities against MCF-7 cells with IC values of 4.98 and 5.85 µM, respectively causing cell death by 97.82 and 97.35%, respectively. Validating the molecular docking studies, both compounds demonstrated promising VEGFR-2 inhibition with IC values of 23.6 and 34.2 µM, compared to Sorafenib (30 µM) and topoisomerase-II inhibition with IC values of 4.1 and 8.6 µM compared to Doxorubicin (9.65 µM). Hence, these two promising compounds could be further tested as effective and selective target-oriented active agents against cancer.
Topics: Humans; DNA Topoisomerases, Type II; Molecular Docking Simulation; Drug Screening Assays, Antitumor; Structure-Activity Relationship; Antineoplastic Agents; Coumarins; Amino Acids; Molecular Structure; Cell Proliferation; Drug Design
PubMed: 36500372
DOI: 10.3390/molecules27238279 -
Nature Communications Dec 2015We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage...
We have previously shown that RNA polymerase II (Pol II) pause release and transcriptional elongation involve phosphorylation of the factor TRIM28 by the DNA damage response (DDR) kinases ATM and DNA-PK. Here we report a significant role for DNA breaks and DDR signalling in the mechanisms of transcriptional elongation in stimulus-inducible genes in humans. Our data show the enrichment of TRIM28 and γH2AX on serum-induced genes and the important function of DNA-PK for Pol II pause release and transcriptional activation-coupled DDR signalling on these genes. γH2AX accumulation decreases when P-TEFb is inhibited, confirming that DDR signalling results from transcriptional elongation. In addition, transcriptional elongation-coupled DDR signalling involves topoisomerase II because inhibiting this enzyme interferes with Pol II pause release and γH2AX accumulation. Our findings propose that DDR signalling is required for effective Pol II pause release and transcriptional elongation through a novel mechanism involving TRIM28, DNA-PK and topoisomerase II.
Topics: Chromatin Immunoprecipitation; Comet Assay; DNA Breaks; DNA Damage; DNA Topoisomerases, Type II; DNA-Activated Protein Kinase; Fluorescent Antibody Technique; HEK293 Cells; Histones; Humans; Microscopy, Confocal; Nuclear Proteins; Phosphorylation; Positive Transcriptional Elongation Factor B; RNA Polymerase II; Real-Time Polymerase Chain Reaction; Repressor Proteins; Signal Transduction; Transcription Elongation, Genetic; Transcription, Genetic; Tripartite Motif-Containing Protein 28
PubMed: 26671524
DOI: 10.1038/ncomms10191 -
The EMBO Journal Jan 2021The juxtaposition of intracellular DNA segments, together with the DNA-passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which...
The juxtaposition of intracellular DNA segments, together with the DNA-passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.
Topics: Adenosine Triphosphatases; Cell Cycle; Cell Cycle Proteins; Chromatids; Chromatin; Chromosomal Proteins, Non-Histone; DNA; DNA Topoisomerases, Type II; DNA-Binding Proteins; Multiprotein Complexes; Saccharomyces cerevisiae; Cohesins
PubMed: 33155682
DOI: 10.15252/embj.2020105393 -
Cellular and Molecular Life Sciences :... Jan 2020The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA... (Review)
Review
The compaction of DNA and the continuous action of DNA transactions, including transcription and DNA replication, create complex DNA topologies that require Type IIA Topoisomerases, which resolve DNA topological strain and control genome dynamics. The human TOP2 enzymes catalyze their reactions via formation of a reversible covalent enzyme DNA-protein crosslink, the TOP2 cleavage complex (TOP2cc). Spurious interactions of TOP2 with DNA damage, environmental toxicants and chemotherapeutic "poisons" perturbs the TOP2 reaction cycle, leading to an accumulation of DNA-protein crosslinks, and ultimately, genomic instability and cell death. Emerging evidence shows that TOP2-DNA protein crosslink (DPC) repair entails multiple strand break repair activities, such as removal of the poisoned TOP2 protein and rejoining of the DNA ends through homologous recombination (HR) or non-homologous end joining (NHEJ). Herein, we discuss the molecular mechanisms of TOP2-DPC resolution, with specific emphasis on the recently uncovered ZATT-licensed TDP2-catalyzed TOP2-DPC reversal mechanism.
Topics: Aminoacyltransferases; Animals; DNA; DNA Breaks; DNA Repair; DNA Topoisomerases, Type II; Humans; Poly-ADP-Ribose Binding Proteins; Protein Conformation; Sumoylation; Transcription Factors
PubMed: 31728578
DOI: 10.1007/s00018-019-03367-z -
Genome Biology Aug 2016Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta...
BACKGROUND
Type II DNA topoisomerases (TOP2) regulate DNA topology by generating transient double stranded breaks during replication and transcription. Topoisomerase II beta (TOP2B) facilitates rapid gene expression and functions at the later stages of development and differentiation. To gain new insight into the genome biology of TOP2B, we used proteomics (BioID), chromatin immunoprecipitation, and high-throughput chromosome conformation capture (Hi-C) to identify novel proximal TOP2B protein interactions and characterize the genomic landscape of TOP2B binding at base pair resolution.
RESULTS
Our human TOP2B proximal protein interaction network included members of the cohesin complex and nucleolar proteins associated with rDNA biology. TOP2B associates with DNase I hypersensitivity sites, allele-specific transcription factor (TF) binding, and evolutionarily conserved TF binding sites on the mouse genome. Approximately half of all CTCF/cohesion-bound regions coincided with TOP2B binding. Base pair resolution ChIP-exo mapping of TOP2B, CTCF, and cohesin sites revealed a striking structural ordering of these proteins along the genome relative to the CTCF motif. These ordered TOP2B-CTCF-cohesin sites flank the boundaries of topologically associating domains (TADs) with TOP2B positioned externally and cohesin internally to the domain loop.
CONCLUSIONS
TOP2B is positioned to solve topological problems at diverse cis-regulatory elements and its occupancy is a highly ordered and prevalent feature of CTCF/cohesin binding sites that flank TADs.
Topics: Alleles; Animals; Binding Sites; CCCTC-Binding Factor; Cell Cycle Proteins; Chromatin; Chromatin Immunoprecipitation; Chromosomal Proteins, Non-Histone; Chromosomes; DNA Topoisomerases, Type II; DNA, Ribosomal; DNA-Binding Proteins; Genome; Humans; Mice; Poly-ADP-Ribose Binding Proteins; Promoter Regions, Genetic; Protein Binding; Protein Interaction Maps; Proteomics; Repressor Proteins; Transcription, Genetic; Cohesins
PubMed: 27582050
DOI: 10.1186/s13059-016-1043-8 -
International Journal of Molecular... Dec 2021DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems...
DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation.
Topics: Arabidopsis; Arabidopsis Proteins; Chromosome Segregation; Chromosomes, Plant; DNA Breaks, Double-Stranded; DNA Repair; DNA Replication; DNA Topoisomerases, Type II; Gamma Rays; Homologous Recombination; Meiosis; Mitomycin; Mutation
PubMed: 34884922
DOI: 10.3390/ijms222313115 -
Cancer Biology & Therapy Dec 2024Ovarian cancer (OC) is a form of gynecological malignancy that is associated with worse patient outcomes than any other cancer of the female reproductive tract....
Ovarian cancer (OC) is a form of gynecological malignancy that is associated with worse patient outcomes than any other cancer of the female reproductive tract. Topoisomerase II α (TOP2A) is commonly regarded as an oncogene that is associated with malignant disease progression in a variety of cancers, its mechanistic functions in OC have yet to be firmly established. We explored the role of TOP2A in OC through online databases, clinical samples, in vitro and in vivo experiments. And initial analyses of public databases revealed high OC-related TOP2A expression in patient samples that was related to poorer prognosis. This was confirmed by clinical samples in which TOP2A expression was elevated in OC relative to healthy tissue. Kaplan-Meier analyses further suggested that higher TOP2A expression levels were correlated with worse prognosis in OC patients. In vitro, TOP2A knockdown resulted in the inhibition of OC cell proliferation, with cells entering G1 phase arrest and undergoing consequent apoptotic death. In rescue assays, TOP2A was confirmed to regulate cell proliferation and cell cycle through AKT/mTOR pathway activity. Mouse model experiments further affirmed the key role that TOP2A plays as a driver of OC cell proliferation. These data provide strong evidence supporting TOP2A as an oncogenic mediator and prognostic biomarker related to OC progression and poor outcomes. At the mechanistic level, TOP2A can control tumor cell growth via AKT/mTOR pathway modulation. These preliminary results provide a foundation for future research seeking to explore the utility of TOP2A inhibitor-based combination treatment regimens in platinum-resistant recurrent OC patients.
Topics: Animals; Female; Humans; Mice; Carcinoma, Ovarian Epithelial; Cell Proliferation; DNA Topoisomerases, Type II; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases
PubMed: 38445610
DOI: 10.1080/15384047.2024.2325126 -
The EMBO Journal Jun 2022Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA,...
Topoisomerase II (TOP2) unlinks chromosomes during vertebrate DNA replication. TOP2 "poisons" are widely used chemotherapeutics that stabilize TOP2 complexes on DNA, leading to cytotoxic DNA breaks. However, it is unclear how these drugs affect DNA replication, which is a major target of TOP2 poisons. Using Xenopus egg extracts, we show that the TOP2 poisons etoposide and doxorubicin both inhibit DNA replication through different mechanisms. Etoposide induces TOP2-dependent DNA breaks and TOP2-dependent fork stalling by trapping TOP2 behind replication forks. In contrast, doxorubicin does not lead to appreciable break formation and instead intercalates into parental DNA to stall replication forks independently of TOP2. In human cells, etoposide stalls forks in a TOP2-dependent manner, while doxorubicin stalls forks independently of TOP2. However, both drugs exhibit TOP2-dependent cytotoxicity. Thus, etoposide and doxorubicin inhibit DNA replication through distinct mechanisms despite shared genetic requirements for cytotoxicity.
Topics: Animals; DNA; DNA Replication; DNA Topoisomerases, Type II; Doxorubicin; Etoposide; Humans; Poisons; Vertebrates
PubMed: 35578785
DOI: 10.15252/embj.2022110632