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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 -
Science Advances Nov 2022Type II topoisomerases (TOP2) are conserved regulators of chromatin topology that catalyze reversible DNA double-strand breaks (DSBs) and are essential for maintaining... (Review)
Review
Type II topoisomerases (TOP2) are conserved regulators of chromatin topology that catalyze reversible DNA double-strand breaks (DSBs) and are essential for maintaining genomic integrity in diverse dynamic processes such as transcription, replication, and cell division. While controlled TOP2-mediated DSBs are an elegant solution to topological constraints of DNA, DSBs also contribute to the emergence of chromosomal translocations and mutations that drive cancer. The central importance of TOP2 enzymes as frontline chemotherapeutic targets is well known; however, their precise biological functions and impact in cancer development are still poorly understood. In this review, we provide an updated overview of TOP2A and TOP2B in the regulation of chromatin topology and transcription, and discuss the recent discoveries linking TOP2 activities with cancer pathogenesis.
Topics: Humans; Chromatin; DNA; DNA Breaks, Double-Stranded; DNA Topoisomerases, Type II; DNA-Binding Proteins; Neoplasms; Poly-ADP-Ribose Binding Proteins; Transcription, Genetic
PubMed: 36322662
DOI: 10.1126/sciadv.add4920 -
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 Chemical Biology Mar 2022Topoisomerase II (topo II) is essential for disentangling newly replicated chromosomes. DNA unlinking involves the physical passage of one duplex through another and...
Topoisomerase II (topo II) is essential for disentangling newly replicated chromosomes. DNA unlinking involves the physical passage of one duplex through another and depends on the transient formation of double-stranded DNA breaks, a step exploited by frontline chemotherapeutics to kill cancer cells. Although anti-topo II drugs are efficacious, they also elicit cytotoxic side effects in normal cells; insights into how topo II is regulated in different cellular contexts is essential to improve their targeted use. Using chemical fractionation and mass spectrometry, we have discovered that topo II is subject to metabolic control through the TCA cycle. We show that TCA metabolites stimulate topo II activity in vitro and that levels of TCA flux modulate cellular sensitivity to anti-topo II drugs in vivo. Our work reveals an unanticipated connection between the control of DNA topology and cellular metabolism, a finding with ramifications for the clinical use of anti-topo II therapies.
Topics: Antineoplastic Agents; DNA; DNA Topoisomerases, Type II; Topoisomerase II Inhibitors
PubMed: 34529934
DOI: 10.1016/j.chembiol.2021.08.014 -
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 -
Antibiotics (Basel, Switzerland) Jul 2023The extensive use of fluoroquinolones has been consequently accompanied by the emergence of bacterial resistance, which triggers the necessity to discover new compounds.... (Review)
Review
The extensive use of fluoroquinolones has been consequently accompanied by the emergence of bacterial resistance, which triggers the necessity to discover new compounds. Delafloxacin is a brand-new anionic non-zwitterionic fluoroquinolone with some structural particularities that give it attractive proprieties: high activity under acidic conditions, greater in vitro activity against Gram-positive bacteria-even those showing resistance to currently-used fluoroquinolones-and nearly equivalent affinity for both type-II topoisomerases (i.e., DNA gyrase and topoisomerase IV). During phases II and III clinical trials, delafloxacin showed non-inferiority compared to standard-of-care therapy in the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia, which resulted in its approval in 2017 by the Food and Drug Administration for indications. Thanks to its overall good tolerance, its broad-spectrum in vitro activity, and its ease of use, it could represent a promising molecule for the treatment of bacterial infections.
PubMed: 37627661
DOI: 10.3390/antibiotics12081241 -
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 -
BioRxiv : the Preprint Server For... 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 different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II). We observed that topo II was at least ~ 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a ~100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive (~ 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.
PubMed: 37873421
DOI: 10.1101/2023.10.03.560726 -
Biomolecules Aug 2021Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential... (Review)
Review
Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential for life and use the same DNA template making conflicts unavoidable. R-loops, DNA supercoiling, DNA secondary structure, and chromatin-binding proteins are all potential obstacles for processive replication or transcription and pose an even more potent threat to genome integrity when these processes co-occur. It is critical to maintaining high fidelity and processivity of transcription and replication while navigating through a complex chromatin environment, highlighting the importance of defining cellular pathways regulating transcription-replication interaction formation, evasion, and resolution. Here we discuss how transcription influences replication fork stability, and the safeguards that have evolved to navigate transcription-replication interactions and maintain genome integrity in mammalian cells.
Topics: Animals; Chromatin; Chromosomes; DNA; DNA Damage; DNA Replication; DNA Topoisomerases, Type II; DNA-Binding Proteins; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerases; Escherichia coli; Genomic Instability; Humans; Mice; Nucleic Acid Conformation; Nucleotides; Oncogenes; Protein Binding; Reproducibility of Results; Ribonuclease H; Saccharomyces cerevisiae; Stochastic Processes; Transcription, Genetic
PubMed: 34439915
DOI: 10.3390/biom11081249