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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 -
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 -
Biochemical Society Transactions Dec 2021Transcription is regulated and mediated by multiprotein complexes in a chromatin context. Transcription causes changes in DNA topology which is modulated by DNA... (Review)
Review
Transcription is regulated and mediated by multiprotein complexes in a chromatin context. Transcription causes changes in DNA topology which is modulated by DNA topoisomerases, enzymes that catalyse changes in DNA topology via transient breaking and re-joining of one or both strands of the phosphodiester backbone. Mammals have six DNA topoisomerases, this review focuses on one, DNA topoisomerase II beta (TOP2B). In the absence of TOP2B transcription of many developmentally regulated genes is altered. Long genes seem particularly susceptible to the lack of TOP2B. Biochemical studies of the role of TOP2B in transcription regulated by ligands such as nuclear hormones, growth factors and insulin has revealed PARP1 associated with TOP2B and also PRKDC, XRCC5 and XRCC6. Analysis of publicly available databases of protein interactions confirms these interactions and illustrates interactions with other key transcriptional regulators including TRIM28. TOP2B has been shown to interact with proteins involved in chromosome organisation including CTCF and RAD21. Comparison of publicly available Chip-seq datasets reveals the location at which these proteins interact with genes. The availability of resources such as large datasets of protein-protein interactions, e.g. BioGrid and IntAct and protein-DNA interactions such as Chip-seq in GEO enables scientists to extend models and propose new hypotheses.
Topics: Animals; DNA Topoisomerases, Type II; Humans; Poly-ADP-Ribose Binding Proteins; Protein Binding; Transcription, Genetic
PubMed: 34747992
DOI: 10.1042/BST20200454 -
Bioorganic Chemistry Jul 2023The DNA topoisomerase enzymes are widely distributed throughout all spheres of life and are necessary for cell function. Numerous antibacterial and cancer... (Review)
Review
The DNA topoisomerase enzymes are widely distributed throughout all spheres of life and are necessary for cell function. Numerous antibacterial and cancer chemotherapeutic drugs target the various topoisomerase enzymes because of their roles in maintaining DNA topology during DNA replication and transcription. Agents derived from natural products, like anthracyclines, epipodophyllotoxins and quinolones, have been widely used to treat a variety of cancers. A very active field of fundamental and clinical research is the selective targeting of topoisomerase II enzymes for cancer treatment. This thematic review summarizes the recent advances in the anticancer activity of the most potent topoisomerase II inhibitors (anthracyclines, epipodophyllotoxins and fluoroquinolones) their modes of action, and structure-activity relationships (SARs) organized chronologically in the last ten years from 2013 to 2023. The review also highlights the mechanism of action and SARs of promising new topoisomerase II inhibitors.
Topics: Topoisomerase II Inhibitors; Antineoplastic Agents; Antibiotics, Antineoplastic; DNA Topoisomerases, Type II; Podophyllotoxin; Anthracyclines; Topoisomerase I Inhibitors
PubMed: 37094479
DOI: 10.1016/j.bioorg.2023.106548 -
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 -
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 -
Natural Product Reports Apr 2022Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been... (Review)
Review
Covering: January 1995 to June 2021Anthracyclines are glycosylated microbial natural products that harbour potent antiproliferative activities. Doxorubicin has been widely used as an anticancer agent in the clinic for several decades, but its use is restricted due to severe side-effects such as cardiotoxicity. Recent studies into the mode-of-action of anthracyclines have revealed that effective cardiotoxicity-free anthracyclines can be generated by focusing on histone eviction activity, instead of canonical topoisomerase II poisoning leading to double strand breaks in DNA. These developments have coincided with an increased understanding of the biosynthesis of anthracyclines, which has allowed generation of novel compound libraries by metabolic engineering and combinatorial biosynthesis. Coupled to the continued discovery of new congeners from rare Actinobacteria, a better understanding of the biology of and improved production methodologies, the stage is set for the development of novel anthracyclines that can finally surpass doxorubicin at the forefront of cancer chemotherapy.
Topics: Anthracyclines; Antineoplastic Agents; DNA Topoisomerases, Type II; Doxorubicin; Polyketides
PubMed: 34951423
DOI: 10.1039/d1np00059d -
Mini Reviews in Medicinal Chemistry 2022Acridine derivatives have been thoroughly investigated and discovered to have multitarget qualities, inhibiting topoisomerase enzymes that regulate topological changes... (Review)
Review
UNLABELLED
Acridine derivatives have been thoroughly investigated and discovered to have multitarget qualities, inhibiting topoisomerase enzymes that regulate topological changes in DNA and interfering with DNA's vital biological function. This article discusses current progress in the realm of novel 9-substituted acridine heterocyclic compounds, including the structure and structure- activity connection of the most promising molecules. The IC values of the new compounds against several human cancer cell lines will also be presented in the publication. The review also looks into the inhibition of topoisomerase by polycyclic aromatic compounds.
BACKGROUND
Acridine rings can be found in molecules used in many different areas, including industry and medicine. Nowadays, acridines with anti-bacterial activity are of research interest due to decreasing bacterial resistance. Some acridine derivatives showed antimalarial or antiviral activity. Acridine derivatives were also investigated for anti-tumor activity due to the interaction with topoisomerase II and DNA base pairs. Considering these possible uses of acridine derivatives, this work overviewed all significant structure performances for the specific action of these compounds.
OBJECTIVE
The objective of this study is to review the activity of acridines as anti-proliferative agents.
METHODS
This review is designed as acridines acting as topoisomerase I and II inhibitors/ poison, Acridines on the G-quadraplux interaction, Acridines with metal complexes, Acridines with quinacrine scaffold, Acridines with sulphur moiety.
CONCLUSION
Although introduced in the 19 century, acridine derivatives are still of scientific interest. In this review, acridine derivatives with various biological activities (antiparasitic, antiviral, anti-bacterial, and antiproliferative) and their structure-activity relationship analyses are presented. Although several mechanisms of their action are known, the only important are discussed here. It can be concluded that the dominant mechanisms are DNA intercalation and interaction with enzymes.
Topics: Acridines; Antimalarials; Antineoplastic Agents; Antiviral Agents; Coordination Complexes; DNA; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Humans; Poisons; Quinacrine; Structure-Activity Relationship; Sulfur
PubMed: 35546777
DOI: 10.2174/1389557522666220511125744