-
The Biochemical Journal Jan 2018Many cancer type-specific anticancer agents have been developed and significant advances have been made toward precision medicine in cancer treatment. However,... (Review)
Review
Many cancer type-specific anticancer agents have been developed and significant advances have been made toward precision medicine in cancer treatment. However, traditional or nonspecific anticancer drugs are still important for the treatment of many cancer patients whose cancers either do not respond to or have developed resistance to cancer-specific anticancer agents. DNA topoisomerases, especially type IIA topoisomerases, are proved therapeutic targets of anticancer and antibacterial drugs. Clinically successful topoisomerase-targeting anticancer drugs act through topoisomerase poisoning, which leads to replication fork arrest and double-strand break formation. Unfortunately, this unique mode of action is associated with the development of secondary cancers and cardiotoxicity. Structures of topoisomerase-drug-DNA ternary complexes have revealed the exact binding sites and mechanisms of topoisomerase poisons. Recent advances in the field have suggested a possibility of designing isoform-specific human topoisomerase II poisons, which may be developed as safer anticancer drugs. It may also be possible to design catalytic inhibitors of topoisomerases by targeting certain inactive conformations of these enzymes. Furthermore, identification of various new bacterial topoisomerase inhibitors and regulatory proteins may inspire the discovery of novel human topoisomerase inhibitors. Thus, topoisomerases remain as important therapeutic targets of anticancer agents.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; Catalytic Domain; DNA; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA, Neoplasm; Drug Design; Gene Expression; Humans; Molecular Docking Simulation; Molecular Targeted Therapy; Neoplasms; Protein Structure, Secondary; Structure-Activity Relationship; Topoisomerase Inhibitors
PubMed: 29363591
DOI: 10.1042/BCJ20160583 -
Cell Jul 2020The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the...
The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
Topics: Aminoquinolines; Animals; CRISPR-Cas Systems; Cell Line; Cytochrome-B(5) Reductase; DNA Damage; DNA Helicases; DNA Repair; DNA Topoisomerases, Type II; Gene Regulatory Networks; Humans; Mice; Picolinic Acids; RNA, Guide, CRISPR-Cas Systems; Tumor Suppressor Protein p53
PubMed: 32649862
DOI: 10.1016/j.cell.2020.05.040 -
Current Protocols Oct 2021Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA...
Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assay of topoisomerase I activity Basic Protocol 2: Assay of topoisomerase II activity Basic Protocol 3: In vivo determination of topoisomerase covalent complexes using the in vivo complex of enzyme (ICE) assay Support Protocol 1: Preparation of mouse tissue for determination of topoisomerase covalent complexes using the ICE assay Support Protocol 2: Using recombinant topoisomerase standard for absolute quantification of cellular TOP2CC Basic Protocol 4: Quantification of topoisomerase-DNA covalent complexes by RADAR/ELISA: The rapid approach to DNA adduct recovery (RADAR) combined with the enzyme-linked immunosorbent assay (ELISA) Basic Protocol 5: Analysis of protein-DNA covalent complexes by RADAR/Western Support Protocol 3: Adduct-Seq to characterize adducted DNA Support Protocol 4: Nuclear fractionation and RNase treatment to reduce sample complexity Basic Protocol 6: Determination of DNA cleavage by purified topoisomerase I Basic Protocol 7: Determination of inhibitor effects on DNA cleavage by topoisomerase II using a plasmid linearization assay Alternate Protocol: Gel electrophoresis determination of topoisomerase II cleavage.
Topics: Animals; DNA Cleavage; DNA Topoisomerases; DNA Topoisomerases, Type II; DNA, Superhelical; Mice; Plasmids
PubMed: 34606690
DOI: 10.1002/cpz1.250 -
Science (New York, N.Y.) Jun 2023Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT)....
Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.
Topics: Animals; Humans; Mice; Chagas Disease; Cryoelectron Microscopy; DNA Topoisomerases, Type II; Trypanosoma; Topoisomerase II Inhibitors; Triazoles; Trypanosomiasis, African; Drug Evaluation, Preclinical
PubMed: 37384702
DOI: 10.1126/science.adh0614 -
Cell Jun 2015Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both...
Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIβ (Topo IIβ), and knockdown of Topo IIβ attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; CCCTC-Binding Factor; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Early Growth Response Protein 1; Etoposide; Gene Expression Regulation; Genes, fos; Genome-Wide Association Study; Mice; Neurons; Repressor Proteins; Transcriptome
PubMed: 26052046
DOI: 10.1016/j.cell.2015.05.032 -
Nature Communications Oct 2023Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create...
Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin's predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.
Topics: DNA Topoisomerases, Type II; DNA; Chromatin; DNA Topoisomerases, Type I; Eukaryotic Cells
PubMed: 37891161
DOI: 10.1038/s41467-023-42600-z -
International Journal of Molecular... Sep 2018Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one... (Review)
Review
Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and β. Topoisomerase IIβ was first reported in 1987. Here we review the research on DNA topoisomerase IIβ over the 30 years since its discovery.
Topics: Animals; Antineoplastic Agents; Cell Cycle; Cloning, Molecular; DNA Topoisomerases, Type II; DNA, Complementary; Gene Expression; Gene Expression Regulation; History, 20th Century; History, 21st Century; Humans; Intracellular Space; Isoenzymes; Molecular Targeted Therapy; Protein Binding; Protein Transport; Research; Topoisomerase II Inhibitors; Transcriptional Activation
PubMed: 30223465
DOI: 10.3390/ijms19092765 -
Molecules (Basel, Switzerland) Nov 2022Topoisomerase inhibitors have been in use clinically for the treatment of several diseases for decades. Although those enzymes are significant molecular targets in... (Review)
Review
Topoisomerase inhibitors have been in use clinically for the treatment of several diseases for decades. Although those enzymes are significant molecular targets in antibacterial and anticancer chemotherapy very little is known about the possibilities to target fungal topoisomerase II (topo II). Raising concern for the fungal infections, lack of effective drugs and a phenomenon of multidrug resistance underlie a strong need to expand the range of therapeutic options. In this review paper, we discussed the usefulness of fungal topo II as a molecular target for new drug discovery. On the basis of previously published data, we described structural and biochemical differences between fungal and human enzymes as well as a molecular basis of differential sensitivity to known anticancer drugs targeting the latter. This review focuses especially on highlighting the differences that may underlie the selectivity of action of new inhibitors. Distinct sites within fungal topo II in comparison with human counterparts are observed and should be further studied to understand the significance of those sites and their possible usage in design of new drugs.
Topics: Humans; Anti-Bacterial Agents; Antifungal Agents; DNA Topoisomerases, Type II; Topoisomerase Inhibitors
PubMed: 36431868
DOI: 10.3390/molecules27227768 -
Cell cycle responses to Topoisomerase II inhibition: Molecular mechanisms and clinical implications.The Journal of Cell Biology Dec 2023DNA Topoisomerase IIA (Topo IIA) is an enzyme that alters the topological state of DNA and is essential for the separation of replicated sister chromatids and the... (Review)
Review
DNA Topoisomerase IIA (Topo IIA) is an enzyme that alters the topological state of DNA and is essential for the separation of replicated sister chromatids and the integrity of cell division. Topo IIA dysfunction activates cell cycle checkpoints, resulting in arrest in either the G2-phase or metaphase of mitosis, ultimately triggering the abscission checkpoint if non-disjunction persists. These events, which directly or indirectly monitor the activity of Topo IIA, have become of major interest as many cancers have deficiencies in Topoisomerase checkpoints, leading to genome instability. Recent studies into how cells sense Topo IIA dysfunction and respond by regulating cell cycle progression demonstrate that the Topo IIA G2 checkpoint is distinct from the G2-DNA damage checkpoint. Likewise, in mitosis, the metaphase Topo IIA checkpoint is separate from the spindle assembly checkpoint. Here, we integrate mechanistic knowledge of Topo IIA checkpoints with the current understanding of how cells regulate progression through the cell cycle to accomplish faithful genome transmission and discuss the opportunities this offers for therapy.
Topics: Cell Cycle Proteins; DNA Topoisomerases, Type II; G2 Phase Cell Cycle Checkpoints; Mitosis; Topoisomerase II Inhibitors
PubMed: 37955972
DOI: 10.1083/jcb.202209125 -
International Journal of Molecular... Sep 2023Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA... (Review)
Review
Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation.
Topics: Animals; DNA; DNA Replication; DNA Topoisomerases, Type II; DNA-Binding Proteins; Gene Expression Regulation; Mammals; Transcription, Genetic; Humans
PubMed: 37834253
DOI: 10.3390/ijms241914806