Topoisomerase II Research Articles

Kinetic pathways of topology simplification by Type-II topoisomerases in knotted supercoiled DNA.

The topological state of covalently closed, double-stranded DNA is defined by the knot type n}{}K and the linking-number difference n}{}Delta Lk relative to unknotted relaxed DNA. DNA topoisomerases are essential enzymes that control the topology of DNA in all cells. In particular, type-II topoisomerases change both n}{}K and n}{}Delta Lk by a duplex-strand-passage mechanism and have been shown to simplify the topology of DNA to levels below thermal equilibrium at the expense of ATP hydrolysis. It remains a key question how small enzymes are able to preferentially select strand passages that result in topology simplification in much larger DNA molecules. Using numerical simulations, we consider the non-equilibrium dynamics of transitions between topological states n}{}(K,Delta Lk) in DNA induced by type-II topoisomerases. For a biological process that delivers DNA molecules in a given topological state n}{}(K,Delta Lk) at a constant rate we fully characterize the pathways of topology simplification by type-II topoisomerases in terms of stationary probability distributions and probability currents on the network of topological states n}{}(K,Delta Lk). In particular, we observe that type-II topoisomerase activity is significantly enhanced in DNA molecules that maintain a supercoiled state with constant torsional tension. This is relevant for bacterial cells in which torsional tension is maintained by enzyme-dependent homeostatic mechanisms such as DNA-gyrase activity.

CryoEM structures of open dimers of gyrase A in complex with DNA illuminate mechanism of strand passage

Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.

TOPII and chromosome movement help remove interlocks between entangled chromosomes during meiosis.

During the zygotene stage of meiosis, normal progression of chromosome synapsis and homologous recombination frequently lead to the formation of structural interlocks between entangled chromosomes. The persistence of interlocks through to the first meiotic division can jeopardize normal synapsis and occasionally chromosome segregation. However, they are generally removed by pachytene. It has been postulated that interlock removal requires one or more active processes, possibly involving topoisomerase II (TOPII) and/or chromosome movement. However, experimental evidence has been lacking. Analysis of a hypomorphic topII mutant and a meiosis-specific topII RNAi knockdown of Arabidopsis thaliana using immunocytochemistry and structured illumination microscopy (SIM) has now enabled us to demonstrate a role for TOPII in interlock resolution. Furthermore, analysis using a nucleoporin nup136 mutant, which affects chromosome movement, reveals that although TOPII activity is required for the removal of some interlock structures, for others, chromosome movement is also necessary. Thus, our study demonstrates that at least two mechanisms are required to ensure interlock removal.

Novel N-1 substituted fluoroquinolones inhibit human topoisomerase I activity and exhibit anti-proliferative activity.

Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.

Reconstitution of anaphase DNA bridge recognition and disjunction.

Faithful chromosome segregation requires that the sister chromatids be disjoined completely. Defective disjunction can lead to the persistence of histone-free threads of DNA known as ultra-fine bridges (UFBs) that connect the separating sister DNA molecules during anaphase. UFBs arise at specific genomic loci and can only be visualized by detection of associated proteins such as PICH, BLM, topoisomerase IIIα, and RPA. However, it remains unknown how these proteins work together to promote UFB processing. We used a combination of ensemble biochemistry and new single-molecule assays to reconstitute key steps of UFB recognition and processing by these human proteins in vitro. We discovered characteristic patterns of hierarchical recruitment and coordinated biochemical activities that were specific for DNA structures modeling UFBs arising at either centromeres or common fragile sites. Our results describe a mechanistic model for how unresolved DNA replication structures are processed by DNA-structure-specific binding factors in mitosis to prevent pathological chromosome nondisjunction.

Structural insights into the gating of DNA passage by the topoisomerase II DNA-gate

Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. Here we present the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. This structure further implicates Top2’s preference for a left-handed DNA braid and allows the construction of a model representing the initial entry of another DNA duplex into the DNA-gate. Steered molecular dynamics calculations suggests the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate.

The Roles of DNA Topoisomerase IIβ in Transcription

Type IIA topoisomerases allow DNA double helical strands to pass through each other by generating transient DNA double strand breaks βDSBs), and in so doing, resolve torsional strain that accumulates during transcription, DNA replication, chromosome condensation, chromosome segregation and recombination. Whereas most eukaryotes possess a single type IIA enzyme, vertebrates possess two distinct type IIA topoisomerases, Topo IIα and Topo IIβ. Although the roles of Topo IIα, especially in the context of chromosome condensation and segregation, have been well-studied, the roles of Topo IIβ are only beginning to be illuminated. This review begins with a summary of the initial studies surrounding the discovery and characterization of Topo IIβ and then focuses on the insights gained from more recent studies that have elaborated important functions for Topo IIβ in transcriptional regulation.

Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site.

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.

Why Two? On the Role of (A-)Symmetry in Negative Supercoiling of DNA by Gyrase.

Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce negative supercoils into DNA by converting a positive DNA node into a negative node through strand passage: First, it cleaves both DNA strands of a double-stranded DNA, termed the G-segment, and then it passes a second segment of the same DNA molecule, termed the T-segment, through the gap created. As a two-fold symmetric enzyme, gyrase contains two copies of all elements that are key for the supercoiling reaction: The GyrB subunits provide two active sites for ATP binding and hydrolysis. The GyrA subunits contain two C-terminal domains (CTDs) for DNA binding and wrapping to stabilize the positive DNA node, and two catalytic tyrosines for DNA cleavage. While the presence of two catalytic tyrosines has been ascribed to the necessity of cleaving both strands of the G-segment to enable strand passage, the role of the two ATP hydrolysis events and of the two CTDs has been less clear. This review summarizes recent results on the role of these duplicate elements for individual steps of the supercoiling reaction, and discusses the implications for the mechanism of DNA supercoiling.

ZATT, TDP2, and SUMO2: breaking the tie that binds TOP2 to DNA

Type II topoisomerase (TOP2) poisons are widely used chemotherapeutics that work by disrupting the cycle of TOP2-DNA interactions that are required for DNA synthesis, gene expression, and the maintenance of genome integrity.

A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus.

Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.

Synthesis, characterization, computational studies and biological activity evaluation of Cu, Fe, Co and Zn complexes with 2-butanone thiosemicarbazone and 1,10-phenanthroline ligands as anticancer and antibacterial agents.

Mixed-ligand metal (II) (M=Cu, Fe, Co and Zn) complexes containing 2-butanone thiosemicarbazone and 1, 10-phenanthroline have been synthesized and characterized by melting point, FT-IR, 1H-NMR, UV-spectrophotometry and molar conductance measurements. All the complexes were soluble in DMSO and DMF. They were thermally stable with high melting points. The computational studies of the complexes were also performed to assess toxicity potential, bioactivity score prediction and drug likeliness assessment based on various drug filters. All the complexes showed no Veber's violations whereas only Cu complex showed one Lipinski's violation. Almost all synthesized compounds were predicted to have no toxic effects. Some of them showed positive bioactivity as enzyme inhibitors. Molecular docking of the complexes was also performed against ribonucleotide diphosphate reductase (RR) and topoisomerase II (Topo II) for minimum binding energy (kcal/mol) calculations. Cu complex was found to have minimum binding energy (-101.13 kcal/mol) released on interaction with Topo II showing a high affinity towards the enzyme, whereas Fe complex had the lowest binding energy (-99.8349 kcal/mol) when docked with RR. The results were compared with two standard drugs i.e. doxorubicin HCl and tetracycline. The ligand was tested for its potential anticancer activity against MDA-MB-231 cell line using MTT assay. Antibacterial activity of the complexes was tested against Staphylococcus aureus and Escherichia coli using the disc diffusion method. Cu (II) complex showed maximum activity against the MDA cells and also exhibited mild antibacterial activity against S. aureus.

Mitochondrial tyrosyl-DNA phosphodiesterase 2 and its TDP2S short isoform.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.

ATP-Dependent Topology Discrimination by Type IIA Topoisomerases: Implications for below Equilibrium Topology Simplification

Type IIA topoisomerases (Topo IIA) are ubiquitous enzymes that play essential roles in maintaining topological homeostasis and in unlinking chromosomes prior to cell division. Topo IIA enzymes, with the exception of DNA gyrase, regulate DNA topology by removing excess DNA supercoils, and inter- and intra-molecular links, corresponding to catenanes and knots respectively. All Topo IIA utilize ATP for their shared catalytic activity in which the enzyme introduces a transient double-strand break in one segment of DNA and transfers a second segment of DNA through this break. The strand passage reaction is coordinated, and made irreversible, through the hydrolysis of ATP. The hydrolysis energy is also coupled to below equilibrium topology simplification activity of Topo IIA that results in the reduction of supercoiling, linking, and knotting to below equilibrium levels. The mechanism of this below equilibrium relaxation remains speculative. In this study, we investigated the correlation between topology-dependent DNA binding and the level of DNA supercoil reduction for two Topo IIA enzymes that exhibit different levels of below equilibrium relaxation: E. coli Topo IV and yeast Topo II. To determine the effect of ATP on the selection of DNA topology, we measured the topology-dependent binding in the presence of two non-hydrolysable ATP analogues: ATPγS and AMPPNP. Topo IV, but not yeast topo II, shows a weak topology-dependent binding in the absence of ATP analogs. Both enzymes exhibit significantly enhanced DNA topology-dependent binding in the presence of ATPγS and AMPPNP. Simulations of Topo IIA activity that incorporate the measured DNA topology dependent binding affinities indicate that DNA topology dependent binding may be a contributing factor explaining non-equilibrium simplification by type IIA topoisomerases.

Kinetic Pathways of Topology Simplification by Type-II Topoisomerases in Knotted, Supercoiled DNA

The topological state of covalently closed, double-stranded DNA is defined by the knot type, K, and the linking number difference from relaxed DNA, ΔLk. DNA topoisomerases are essential enzymes that regulate topological states of DNA in vivo: type-I topoisomerases (topo-Is) change ΔLk, thereby regulating the torsional tension, whereas type-II topoisomerases (topo-IIs) change both (ΔLk, K) by passing one DNA helix through another. A critical biological function of type-II enzymes is the elimination of knots in DNA because their presence impedes transcription and replication. It has been a long-standing puzzle how small type-II enzymes select passages that unknot large DNA molecules, since topology is a global property which cannot be determined by local DNA-enzyme interactions. Previous studies addressing this question have focused on the equilibrium distribution P(ΔLk, K). Motivated by the fact that topo-IIs reduce the knotting level below equilibrium at the expense of ATP hydrolysis, we set out to study topoisomerase activity in the framework of non-equilibrium thermodynamics. We consider the dynamics of transitions in a network of topological states (ΔLk, K) induced by type-II and type-I action by solving the master equation for the time-dependent probability distribution P(ΔLk, K; t). We fully characterize non-equilibrium steady states generated by injecting DNA molecules in a given topological state in terms of stationary probability distributions and currents in the network. This allows us, for the first time, to predict detailed kinetic pathways of topoisomerase action as a function of geometry of the enzyme. In particular, we find that unknotting activity of topo-II is significantly enhanced in DNA molecules which maintain a supercoiled state with constant torsional tension; this is relevant for bacterial cells in which the torsional tension is maintained by a homeostatic mechanism using topo I and DNA gyrase.

Topoisomerases as anticancer targets.

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.

DNA Topoisomerases as Targets for Antibacterial Agents.

DNA topoisomerases are proven therapeutic targets of antibacterial agents. Quinolones, especially fluoroquinolones, are the most successful topoisomerase-targeting antibacterial drugs. These drugs target type IIA topoisomerases in bacteria. Recent structural and biochemical studies on fluoroquinolones have provided the molecular basis for both their mechanism of action, as well as the molecular basis of bacterial resistance. Due to the development of drug resistance, including fluoroquinolone resistance, among bacterial pathogens, there is an urgent need to discover novel antibacterial agents. Recent advances in topoisomerase inhibitors may lead to the development of novel antibacterial drugs that are effective against fluoroquinolone-resistant pathogens. They include type IIA topoisomerase inhibitors that either interact with the GyrB/ParE subunit or form nick-containing ternary complexes. In addition, several topoisomerase I inhibitors have recently been identified. Thus, DNA topoisomerases remain important targets of antibacterial agents.

New multiplex PCR for species-specific diagnosis of human candidiasis

Candidiases is a group of opportunistic infections caused by yeasts belonging to the genus Candida. Candida albicans is the most prevalent species in both superficial and deep infections, however, the clinical importance of non-albicans Candida has increased during the last decade, driving an urgent need for diagnostic tests that allow for species-level resolution and selection of the optimum therapeutic approach. To design and to optimize a new multiplex PCR assay for the simultaneous identification of the five most relevant species of Candida involved in human candidiasis etiology. For primers design, the physical and thermodynamic restrictions that affect multiplex PCR performance were analyzed using Gene Runner and Mult-PSOS. As templates, the internal transcribed region 2 (ITR2) was selected for C. albicans (AJ249486.1), and topoisomerase II (TOPII) for C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1), and C. guillermondii (AB049145.1). We used ATCC strains of all these five species and clinical isolates as templates. We designed ten oligonucleotides for the simultaneous amplification of the Candida species. The electrophoresis band profile was: C. albicans (206 bp), C. guillermondii (244 bp), C. tropicalis (474 bp), C. parasilopsis (558 bp), and C. krusei (419 bp). The new multiplex PCR assay designed in this study allowed a simultaneous and efficient amplification of the amplicons corresponding to the five species of Candida under study, with an adequate resolution in standard agarose gel.

Discovery of substituted oxadiazoles as a novel scaffold for DNA gyrase inhibitors

DNA gyrase and topoisomerase IV are type IIa topoisomerases that are essential bacterial enzymes required to oversee the topological state of DNA during transcription and replication processes. Their ATPase domains, GyrB and ParE, respectively, are recognized as viable targets for small molecule inhibitors, however, no synthetic or natural product GyrB/ParE inhibitors have so far reached the clinic for use as novel antibacterial agents, except for novobiocin which was withdrawn from the market. In the present study, a series of substituted oxadiazoles have been designed and synthesized as potential DNA gyrase inhibitors. Structure-based optimization resulted in the identification of compound 35, displaying an IC50 of 1.2 μM for Escherichia coli DNA gyrase, while also exhibiting a balanced low micromolar inhibition of E. coli topoisomerase IV and of the respective Staphylococcus aureus homologues. The most promising inhibitors identified from each series were ultimately evaluated against selected Gram-positive and Gram-negative bacterial strains, of which compound 35 inhibited Enterococcus faecalis with a MIC90 of 75 μM. Our study thus provides further insight into the structural requirements of substituted oxadiazoles for dual inhibition of DNA gyrase and topoisomerase IV.

Expression of excision repair cross-complementing 1, topoisomeraseII, ribonucleotide reductase M1, β3-tubulin and thymidylate synthase in lung cancer

Objective To analyze the expression characteristics of excision repair cross-complementing 1(ERCC1), topoisomerase Ⅱ (TOPO Ⅱ), ribonucleotide reductase M1(RRM1), β3-tubulin and thymidylate synthase (TS) in lung cancer and their associations with the pathological types. Methods The immunohistochemical EnVision method was used to determine the expression of ERCC1, TOPOⅡ, RRM1, β3-tubulin and TS in 548 patients who were diagnosed as lung cancer from January 2011 to December 2014. Variance analysis was performed to analyze their expression characteristics among different pathological types and correlation. Results The expression positive rates of ERCC1, TOPOⅡ, RRM1, β3-tubulin and TS were 61.86% (339/548), 91.06% (499/548), 62.59% (343/548), 73.18% (401/548) and 70.44% (386/548), respectively. The expression of ERCC1 was weak positive mostly (P<0.05), meanwhile the expression of TOPO Ⅱ was medium-strong positive mostly (P<0.05). In ERCC1 group, the positive rate of squamous cell carcinoma was higher than that of adenocarcinoma [57.39% (167/291) vs. 42.61% (124/291), P=0.000]. In weak positive of TOPOⅡ group, the proportion of adenocarcinoma was higher than that of squamous cell carcinoma[23.58% (100/137) vs. 8.73% (37/137), P=0.000]. In medium-strong positive of TOPO Ⅱ group, the proportion of squamous cell carcinoma was higher than that of adenocarcinoma[47.41% (201/287/) vs. 20.28% (86/287), P=0.000]. The expressions of ERCC1, TOPO Ⅱ, RRM1, β3-tubulin and TS were irrelevant (r=0.4, P=0.397). Conclusions The expressions of ERCC1 and TOPO Ⅱ are higher in squamous cell carcinoma than those in adenocarcinoma. The expression of ERCC1 is weak positive mostly, meanwhile the expression of TOPO Ⅱ is medium-strong positive mostly. There is no correlation between them. Key words: Lung neoplasms; Excision repair cross-complementing 1; Topoisomerase Ⅱ; Thymidylate synthase