ABSTRACT
Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
Subject(s)
DNA, Mitochondrial , Mitochondria , Humans , Mitochondria/metabolism , DNA, Mitochondrial/metabolism , DNA Topoisomerases, Type I/metabolism , DNA Replication/genetics , DNA Topoisomerases/geneticsABSTRACT
BACKGROUND: Hoffman syndrome is a syndromic, inborn error of immunity due to autosomal-dominant mutations in TOP2B, an essential gene required to alleviate topological stress during DNA replication and gene transcription. Although mutations identified in patients lead to a block in B-cell development and the absence of circulating B cells, an effect on natural killer (NK) cells was not previously examined. OBJECTIVE: We sought to determine whether disease-associated mutations in TOP2B impact NK-cell development and function. METHODS: Using a knockin murine model and patient-derived induced pluripotent stem cells (iPSCs), we investigated NK-cell development in mouse bone marrow and spleen, and performed immunophenotyping by flow cytometry, gene expression, and functional assessment of cytotoxic activity in murine NK cells, and human IPSC-derived NK cells. RESULTS: Mature NK cells were reduced in the periphery of TOP2B knockin mice consistent with patient reports, with reduced cytotoxicity toward target cell lines. IPSCs were successfully derived from patients with Hoffman syndrome, but under optimal conditions showed reduced cytotoxicity compared with iPSC-derived NK cells from healthy controls. CONCLUSIONS: Hoffman syndrome-associated mutations in TOP2B impact NK-cell development and function in murine and human models.
Subject(s)
Induced Pluripotent Stem Cells , Killer Cells, Natural , Animals , Cell Line , Craniofacial Abnormalities , Humans , Induced Pluripotent Stem Cells/metabolism , Limb Deformities, Congenital , Mice , Mutation , Primary Immunodeficiency Diseases , Urogenital AbnormalitiesABSTRACT
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.
Subject(s)
DNA Topoisomerases, Type II , Transcription, Genetic , Animals , DNA , DNA Replication , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Mammals/metabolism , HumansABSTRACT
DNA topoisomerases regulate the topological state of DNA, relaxing DNA supercoils and resolving catenanes and knots that result from biologic processes, such as transcription and replication. DNA topoisomerase II (TOP2) enzymes achieve this by binding DNA and introducing an enzyme-bridged DNA double-strand break (DSB) where each protomer of the dimeric enzyme is covalently attached to the 5' end of the cleaved DNA via an active site tyrosine phosphodiester linkage. The enzyme then passes a second DNA duplex through the DNA break, before religation and release of the enzyme. However, this activity is potentially hazardous to the cell, as failure to complete religation leads to persistent TOP2 protein-DNA covalent complexes, which are cytotoxic. Indeed, this property of topoisomerase has been exploited in cancer therapy in the form of topoisomerase poisons which block the religation stage of the reaction cycle, leading to an accumulation of topoisomerase-DNA adducts. A number of parallel cellular processes have been identified that lead to removal of these covalent TOP2-DNA complexes, facilitating repair of the resulting protein-free DSB by standard DNA repair pathways. These pathways presumably arose to repair spontaneous stalled or poisoned TOP2-DNA complexes, but understanding their mechanisms also has implications for cancer therapy, particularly resistance to anti-cancer TOP2 poisons and the genotoxic side effects of these drugs. Here, we review recent progress in the understanding of the processing of TOP2 DNA covalent complexes, the basic components and mechanisms, as well as the additional layer of complexity posed by the post-translational modifications that modulate these pathways. SIGNIFICANCE STATEMENT: Multiple pathways have been reported for removal and repair of TOP2-DNA covalent complexes to ensure the timely and efficient repair of TOP2-DNA covalent adducts to protect the genome. Post-translational modifications, such as ubiquitination and SUMOylation, are involved in the regulation of TOP2-DNA complex repair. Small molecule inhibitors of these post-translational modifications may help to improve outcomes of TOP2 poison chemotherapy, for example by increasing TOP2 poison cytotoxicity and reducing genotoxicity, but this remains to be determined.
Subject(s)
DNA Repair/physiology , DNA Topoisomerases, Type II/metabolism , Topoisomerase II Inhibitors/pharmacology , DNA Breaks/drug effects , DNA Damage/drug effects , DNA Damage/physiology , DNA Repair/drug effects , DNA Topoisomerases, Type II/genetics , Humans , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/physiologyABSTRACT
DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double-strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct, which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase that utilizes energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A and TOP2B complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2A.X, indicative of fewer DSBs. This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. SIGNIFICANCE STATEMENT: TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. A better understanding of how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.
Subject(s)
Acetanilides/pharmacology , Benzothiazoles/pharmacology , DNA Topoisomerases, Type II/metabolism , DNA/metabolism , Etoposide/pharmacology , Poly-ADP-Ribose Binding Proteins/metabolism , Valosin Containing Protein/metabolism , Adenosine Triphosphate/metabolism , Gene Knockdown Techniques , Histones/metabolism , Humans , Hydrolysis , K562 Cells , Phosphorylation , Protein Folding , Protein Stability , Valosin Containing Protein/geneticsABSTRACT
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.
Subject(s)
DNA Topoisomerases, Type II/physiology , Poly-ADP-Ribose Binding Proteins/physiology , Transcription, Genetic/physiology , Animals , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Humans , Poly-ADP-Ribose Binding Proteins/genetics , Poly-ADP-Ribose Binding Proteins/metabolism , Protein BindingABSTRACT
Type II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational 'landscape' of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2ß) is additionally revealed to be significantly regulated by the enzymes' adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.
Subject(s)
Ciprofloxacin/pharmacology , DNA Topoisomerases, Type II/metabolism , Naphthyridines/pharmacology , Saccharomyces cerevisiae Proteins/metabolism , Thiazoles/pharmacology , Topoisomerase II Inhibitors/pharmacology , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Drug Resistance/drug effects , Drug Resistance/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Models, Molecular , Mutation , Nucleic Acid Conformation , Protein Binding , Protein Domains , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.
Subject(s)
DNA Topoisomerases, Type II/metabolism , Nucleosides/pharmacology , Poly-ADP-Ribose Binding Proteins/metabolism , Sulfonamides/pharmacology , Ubiquitin-Activating Enzymes/antagonists & inhibitors , Cell Line , DNA/metabolism , DNA Topoisomerases, Type II/chemistry , Humans , K562 Cells , Poly-ADP-Ribose Binding Proteins/chemistry , Proteasome Endopeptidase Complex/metabolism , Proteolysis/drug effects , Pyrazoles , Pyrimidines , Sulfides , Ubiquitin/metabolism , Ubiquitination/drug effectsABSTRACT
Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase IIß (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.
Subject(s)
Anthracyclines/pharmacology , DNA Adducts/metabolism , DNA Topoisomerases, Type II/metabolism , Etoposide/pharmacology , Mitoxantrone/pharmacology , Cardiotoxicity , Cell Line, Tumor , Cell Survival/drug effects , DNA Adducts/drug effects , Dose-Response Relationship, Drug , Doxorubicin/adverse effects , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/drug effects , K562 Cells , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Topoisomerase II Inhibitors/pharmacologyABSTRACT
2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here, we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (TOP2B) covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide, which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination since both occur even under conditions of depleted ubiquitin. Like etoposide, since PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.
Subject(s)
Aminopyridines/pharmacology , Aminopyridines/poisoning , DNA Topoisomerases, Type II/biosynthesis , Deubiquitinating Enzymes/antagonists & inhibitors , Deubiquitinating Enzymes/metabolism , Enzyme Inhibitors/pharmacology , Poly-ADP-Ribose Binding Proteins/biosynthesis , Thiocyanates/pharmacology , Thiocyanates/poisoning , HeLa Cells , Humans , K562 Cells , Poly-ADP-Ribose Binding Proteins/agonistsABSTRACT
DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5'-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5' tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition.
Subject(s)
DNA Repair , DNA Topoisomerases, Type II/metabolism , DNA/metabolism , Nuclear Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Transcription Factors/metabolism , DNA/genetics , DNA Adducts/genetics , DNA Adducts/metabolism , DNA Breaks, Double-Stranded/drug effects , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins , Etoposide/pharmacology , Humans , K562 Cells , Nuclear Proteins/genetics , Phosphoric Diester Hydrolases , Poly-ADP-Ribose Binding Proteins/antagonists & inhibitors , Poly-ADP-Ribose Binding Proteins/genetics , RNA Interference , Sumoylation , Topoisomerase II Inhibitors/pharmacology , Transcription Factors/geneticsABSTRACT
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 ß. Topoisomerase IIß was first reported in 1987. Here we review the research on DNA topoisomerase IIß over the 30 years since its discovery.
Subject(s)
DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , Research , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Cycle/genetics , Cloning, Molecular , DNA Topoisomerases, Type II/chemistry , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression , Gene Expression Regulation , History, 20th Century , History, 21st Century , Humans , Intracellular Space/metabolism , Isoenzymes , Molecular Targeted Therapy , Protein Binding , Protein Transport , Research/history , Topoisomerase II Inhibitors/pharmacology , Topoisomerase II Inhibitors/therapeutic use , Transcriptional ActivationABSTRACT
Myeloperoxidase is expressed exclusively in granulocytes and immature myeloid cells and transforms the topoisomerase II (TOP2) poisons etoposide and mitoxantrone to chemical forms that have altered DNA damaging properties. TOP2 poisons are valuable and widely used anticancer drugs, but they are associated with the occurrence of secondary acute myeloid leukemias. These factors have led to the hypothesis that myeloperoxidase inhibition could protect hematopoietic cells from TOP2 poison-mediated genotoxic damage and, therefore, reduce the rate of therapy-related leukemia. We show here that myeloperoxidase activity leads to elevated accumulation of etoposide- and mitoxantrone-induced TOP2A and TOP2B-DNA covalent complexes in cells, which are converted to DNA double-strand breaks. For both drugs, the effect of myeloperoxidase activity was greater for TOP2B than for TOP2A. This is a significant finding because TOP2B has been linked to genetic damage associated with leukemic transformation, including etoposide-induced chromosomal breaks at the MLL and RUNX1 loci. Glutathione depletion, mimicking in vivo conditions experienced during chemotherapy treatment, elicited further MPO-dependent increase in TOP2A and especially TOP2B-DNA complexes and DNA double-strand break formation. Together these results support targeting myeloperoxidase activity to reduce genetic damage leading to therapy-related leukemia, a possibility that is enhanced by the recent development of novel specific myeloperoxidase inhibitors for use in inflammatory diseases involving neutrophil infiltration.
Subject(s)
Antineoplastic Agents/therapeutic use , Cytoprotection/drug effects , DNA Damage , Etoposide/pharmacology , Mitoxantrone/pharmacology , Myeloid Cells/pathology , Neoplasms/drug therapy , Peroxidase/metabolism , Antigens, Neoplasm/metabolism , Cell Line, Tumor , DNA/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Glutathione/metabolism , Histones/metabolism , Humans , Myeloid Cells/drug effects , Neoplasms/enzymology , Neoplasms/pathology , Peroxidase/antagonists & inhibitors , Phosphorylation/drug effects , Poly-ADP-Ribose Binding Proteins , Small Molecule Libraries/pharmacologyABSTRACT
To elucidate one or more mechanisms through which microrchidia (MORC) proteins impact immunity, epigenetic gene silencing, and DNA modifications, the enzymatic activities of plant MORCs were characterized. Previously, we showed that plant MORC1s have ATPase and DNA endonuclease activities. Here, we demonstrate that plant MORCs have topoisomerase type II (topo II)-like activities, as they i) covalently bind DNA, ii) exhibit DNA-stimulated ATPase activity, iii) relax or nick supercoiled DNA, iv) catenate DNA, and v) decatenante kinetoplast DNA. Mutational analysis of tomato SlMORC1 suggests that a K loop-like sequence is required to couple DNA binding to ATPase stimulation as well as for efficient SlMORC1's DNA relaxation and catenation activities and in planta suppression of INF1-induced cell death, which is related to immunity. Human MORCs were found to exhibit the same topo II-like DNA modification activities as their plant counterparts. In contrast to typical topo IIs, SlMORC1 appears to require one or more accessory factors to complete some of its enzymatic activities, since addition of tomato extracts were needed for ATP-dependent, efficient conversion of supercoiled DNA to nicked/relaxed DNA and catenanes and for formation of topoisomer intermediates. Both plant and human MORCs bind salicylic acid; this suppresses their decatenation but not relaxation activity.
Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA, Superhelical/metabolism , Nuclear Proteins/metabolism , Plant Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Biocatalysis , DNA/metabolism , Humans , Hydrolysis , Lysine/metabolism , Mutation/genetics , Nuclear Proteins/chemistry , Plant Extracts/metabolism , Plant Proteins/chemistry , Protein Binding , Salicylic Acid/metabolismABSTRACT
TOP2A and TOP2B are type II topoisomerase enzymes that have important but distinct roles in DNA replication and RNA transcription. Recently, TOP2B has been implicated in the transcription of long genes in particular that play crucial roles in neural development and are susceptible to mutations contributing to neurodevelopmental conditions such as autism and schizophrenia. This study maps their expression in the early foetal human telencephalon between 9 and 12 post-conceptional weeks. TOP2A immunoreactivity was restricted to cell nuclei of the proliferative layers of the cortex and ganglionic eminences (GE), including the ventricular zone and subventricular zone (SVZ) closely matching expression of the proliferation marker KI67. Comparison with sections immunolabelled for NKX2.1, a medial GE (MGE) marker, and PAX6, a cortical progenitor cell and lateral GE (LGE) marker, revealed that TOP2A-expressing cells were more abundant in MGE than the LGE. In the cortex, TOP2B is expressed in cell nuclei in both proliferative (SVZ) and post-mitotic compartments (intermediate zone and cortical plate) as revealed by comparison with immunostaining for PAX6 and the post-mitotic neuron marker TBR1. However, co-expression with KI67 was rare. In the GE, TOP2B was also expressed by proliferative and post-mitotic compartments. In situ hybridisation studies confirmed these patterns of expression, except that TOP2A mRNA is restricted to cells in the G2/M phase of division. Thus, during early development, TOP2A is likely to have a role in cell proliferation, whereas TOP2B is expressed in post-mitotic cells and may be important in controlling expression of long genes even at this early stage.
Subject(s)
Antigens, Neoplasm/biosynthesis , DNA Topoisomerases, Type II/biosynthesis , DNA-Binding Proteins/biosynthesis , Fetus/enzymology , Neurogenesis/physiology , Telencephalon/embryology , Telencephalon/enzymology , Humans , Immunohistochemistry , In Situ Hybridization , Poly-ADP-Ribose Binding ProteinsABSTRACT
Pluripotent-specific inhibitors (PluriSIns) make a powerful tool to study the mechanisms controlling the survival of human pluripotent stem cells (hPSCs). Here, we characterize the mechanism of action of PluriSIn#2, a compound that selectively eliminates undifferentiated hPSCs, while sparing various other cell types derived from them. Toxicogenomic analysis predicts this compound to be a topoisomerase inhibitor. Gene expression analyses reveal that one of the human topoisomerase enzymes, topoisomerase II alpha (TOP2A), is uniquely expressed in hPSCs: TOP2A is highly expressed in undifferentiated cells, is downregulated during their differentiation, and its expression depends on the expression of core pluripotency transcription factors. Furthermore, siRNA-based knockdown of TOP2A in undifferentiated hPSCs results in their cell death, revealing that TOP2A expression is required for the survival of these cells. We find that PluriSIn#2 does not directly inhibit TOP2A enzymatic activity, but rather selectively represses its transcription, thereby significantly reducing TOP2A protein levels. As undifferentiated hPSCs require TOP2A activity for their survival, TOP2A inhibition by PluriSIn#2 thus causes their cell death. Therefore, TOP2A dependency can be harnessed for the selective elimination of tumorigenic hPSCs from culture.
Subject(s)
DNA-Binding Proteins/antagonists & inhibitors , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Topoisomerase Inhibitors/pharmacology , Antigens, Neoplasm/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/enzymology , Humans , Pluripotent Stem Cells/enzymology , Poly-ADP-Ribose Binding Proteins , Small Molecule Libraries/pharmacologyABSTRACT
Topoisomerase poisons such as the epipodophyllotoxin etoposide are widely used effective cytotoxic anticancer agents. However, they are associated with the development of therapy-related acute myeloid leukemias (t-AMLs), which display characteristic balanced chromosome translocations, most often involving the mixed lineage leukemia (MLL) locus at 11q23. MLL translocation breakpoints in t-AMLs cluster in a DNase I hypersensitive region, which possesses cryptic promoter activity, implicating transcription as well as topoisomerase II activity in the translocation mechanism. We find that 2-3% of MLL alleles undergoing transcription do so in close proximity to one of its recurrent translocation partner genes, AF9 or AF4, consistent with their sharing transcription factories. We show that most etoposide-induced chromosome breaks in the MLL locus and the overall genotoxicity of etoposide are dependent on topoisomerase IIß, but that topoisomerase IIα and -ß occupancy and etoposide-induced DNA cleavage data suggest factors other than local topoisomerase II concentration determine specific clustering of MLL translocation breakpoints in t-AML. We propose a model where DNA double-strand breaks (DSBs) introduced by topoisomerase IIß into pairs of genes undergoing transcription within a common transcription factory become stabilized by antitopoisomerase II drugs such as etoposide, providing the opportunity for illegitimate end joining and translocation.
Subject(s)
DNA Breaks, Double-Stranded , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Leukemia, Myeloid, Acute/genetics , Models, Biological , Myeloid-Lymphoid Leukemia Protein/genetics , Neoplasms, Second Primary/genetics , Translocation, Genetic/genetics , Chromatin Immunoprecipitation , Etoposide , Fluorescence , Humans , In Situ Hybridization, Fluorescence , Micronucleus TestsABSTRACT
Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment.
Subject(s)
Chromosome Breakage , Core Binding Factor Alpha 2 Subunit/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogenes/genetics , Transcription Factors/genetics , Translocation, Genetic , Antineoplastic Agents/adverse effects , Base Sequence , Cell Line, Tumor , Chromatin/metabolism , DNA Breaks, Double-Stranded , DNA Topoisomerases, Type II/genetics , Etoposide/adverse effects , Humans , MDS1 and EVI1 Complex Locus Protein , Molecular Sequence Data , Poly-ADP-Ribose Binding Proteins , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/chemically induced , RUNX1 Translocation Partner 1 ProteinABSTRACT
Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and ß), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIß revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.
Subject(s)
Biocatalysis , DNA Cleavage/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Salicylic Acid/pharmacology , Topoisomerase II Inhibitors/pharmacology , Antigens, Neoplasm/metabolism , Cell Line, Tumor , DNA/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , SalicylatesABSTRACT
Because of their essentiality for DNA replication, transcription, and repair, type II topoisomerases are targets for antibacterial and anticancer drugs. There are two type II topoisomerases in humans, topoisomerase IIα (TOP2A) and topoisomerase IIß (TOP2B), and two in bacteria, gyrase and topoisomerase IV. Inhibition of one or both of the human type II topoisomerases by antibacterial compounds targeting their bacterial counterparts could result in toxicity. In addition, side effects of anticancer drugs targeting TOP2A could result from inhibition of TOP2B. A simple and rapid biochemical assay for the activity of TOP2A and TOP2B would be advantageous for screening for novel inhibitors, testing them for selectivity for one enzyme over the other, and testing for potential toxicity of antibacterial type II topoisomerases mediated by human topoisomerase II inhibition. In this paper, we show that a previously reported high-throughput, fluorescence anisotropy-based assay for ATP-dependent relaxation of supercoiled DNA by human TOP2A can also be used under identical conditions for human TOP2B. We used this assay to compare the potencies versus both enzymes of 19 compounds reported in the literature to inhibit human and/or bacterial type II topoisomerases. We also used the assay to investigate the effect of ATP concentration on inhibitor potencies.