Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life.

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

Characterization and antimicrobial activity of endophytic fungi from medicinal plant Agave americana.

Endophytic microorganisms associated with medicinal plants are of particular interest as they are a potential source of new bioactive chemicals effective against novel emerging and drug-resistant pathogens. Agave americana is a tropical medicinal plant with antibacterial, antifungal, and anticancer properties. We studied the biodiversity of fungal endophytes of A. americana and their antimicrobial production potential. Isolated endophytic fungi were classified into 32 morphotypes (15 from stem and 17 from leaf) based on their cultural and morphological characteristics. Among the fungal crude extracts tested, 82% of isolates from the leaves and 80% of the isolates from the stem showed antibacterial activity against the bacterial strains (Escherichia coli ATTC 25902, Staphylococcus aureus ATTC 14775, and Bacillus subtilis NRRL 5109) tested. Extracts from four fungal isolates from leaves showed antifungal activity against at least one of the fungal strains (Candida albicans ATTC 10231 and Aspergillus fumigatus NRRL 5109) tested. Crude extracts of seven fungal isolates showed a zone of inhibition of more than 11 mm at 10 mgml-1 against both Gram-positive and Gram-negative bacteria tested. Penicillium, Colletotrichum, Curvularia, Pleosporales, Dothideomycetes, and Pleurotus are the main endophytes responsible for bioactive potential. These results indicate that A. americana harbors endophytes capable of producing antimicrobial metabolites.

The interaction between transport-segment DNA and topoisomerase IA-crystal structure of MtbTOP1 in complex with both G- and T-segments.

Each catalytic cycle of type IA topoisomerases has been proposed to comprise multistep reactions. The capture of the transport-segment DNA (T-segment) into the central cavity of the N-terminal toroidal structure is an important action, which is preceded by transient gate-segment (G-segment) cleavage and succeeded by G-segment religation for the relaxation of negatively supercoiled DNA and decatenation of DNA. The T-segment passage in and out of the central cavity requires significant domain-domain rearrangements, including the movement of D3 relative to D1 and D4 for the opening and closing of the gate towards the central cavity. Here we report a direct observation of the interaction of a duplex DNA in the central cavity of a type IA topoisomerase and its associated domain-domain conformational changes in a crystal structure of a Mycobacterium tuberculosis topoisomerase I complex that also has a bound G-segment. The duplex DNA within the central cavity illustrates the non-sequence-specific interplay between the T-segment DNA and the enzyme. The rich structural information revealed from the novel topoisomerase-DNA complex, in combination with targeted mutagenesis studies, provides new insights into the mechanism of the topoisomerase IA catalytic cycle.

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4.

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb's toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM).

Proton Motive Force-Disrupting Antimycobacterial Guanylurea Polymer.

Mycobacterial infectious diseases, including tuberculosis (TB), severely threaten global public health. Nonreplicating Mycobacterium tuberculosis (Mtb) is extremely difficult to eradicate using current TB drugs that primarily act on replicating cells. Novel TB drugs acting on unconventional targets are needed to combat TB efficiently. Although membrane-disrupting antimicrobial peptides and their synthetic mimics exhibit the potential to kill persisters, the lack of microbe selectivity, especially toward mycobacteria, has been a concern. Here, we report that the recently developed poly(guanylurea)-piperazine (PGU-P) shows fast and selective mycobactericidal effects. Using a nonpathogenic model organism, Mycobacterium smegmatis (Msm), we have found that the mycobactericidal effects of PGU-P are correlated to the disruption of the mycobacterial membrane potential and bioenergetics. Accordingly, PGU-P also potentiates bedaquiline, an oxidative phosphorylation-targeting TB drug disturbing mycobacterial bioenergetics. Importantly, PGU-P also exhibits a promising activity against pathogenic Mtb with a minimum inhibitory concentration of 37 µg/mL. Our results support that PGU-P is a novel class of antimycobacterial biomaterial, and the unique structural feature can contribute to developing novel antimycobacterial drugs.

Potent Inhibition of Bacterial DNA Gyrase by Digallic Acid and Other Gallate Derivatives.

Bacterial DNA gyrase, an essential enzyme, is a validated target for discovering and developing new antibiotics. Here we screened a pool of polyphenols and discovered that digallic acid is a potent DNA gyrase inhibitor. We also found that several food additives based on gallate, such as dodecyl gallate, potently inhibit bacterial DNA gyrase. Interestingly, the IC50 of these gallate derivatives against DNA gyrase is correlated with the length of hydrocarbon chain connecting to the gallate. These new bacterial DNA gyrase inhibitors are ATP competitive inhibitors of DNA gyrase. Our results also show that digallic acid and certain gallate derivatives potently inhibit E. coli DNA topoisomerase IV. Several gallate derivatives have strong antimicrobial activities against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). This study provides a solid foundation for the design and synthesis of gallate-based DNA gyrase inhibitors that may be used to combat antibacterial resistance.

Microbial Type IA Topoisomerase C-Terminal Domain Sequence Motifs, Distribution and Combination.

Type IA topoisomerases have highly conserved catalytic N-terminal domains for the cleaving and rejoining of a single DNA/RNA strand that have been extensively characterized. In contrast, the C-terminal region has been less covered. Two major types of small tandem C-terminal domains, Topo_C_ZnRpt (containing C4 zinc finger) and Topo_C_Rpt (without cysteines) were initially identified in Escherichia coli and Mycobacterium tuberculosis topoisomerase I, respectively. Their structures and interaction with DNA oligonucleotides have been revealed in structural studies. Here, we first present the diverse distribution and combinations of these two structural elements in various bacterial topoisomerase I (TopA). Previously, zinc fingers have not been seen in type IA topoisomerases from well-studied fungal species within the phylum Ascomycota. In our extended studies of C-terminal DNA-binding domains, the presence of zf-GRF and zf-CCHC types of zinc fingers in topoisomerase III (Top3) from fungi species in many phyla other than Ascomycota has drawn our attention. We secondly analyze the distribution and combination of these fungal zf-GRF- and zf-CCHC-containing domains. Their potential structures and DNA-binding mechanism are evaluated. The highly diverse arrangements and combinations of these DNA/RNA-binding domains in microbial type IA topoisomerase C-terminal regions have important implications for their interactions with nucleic acids and protein partners as part of their physiological functions.

A Yeast-Based Screening System for Differential Identification of Poisons and Suppressors of Human Topoisomerase I.

BACKGROUND: Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern)-induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1-DNA covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poisons developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. METHODS: In this study we set up a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. The effect of test compounds on growth is monitored with and without overexpression of the recombinant human TOP1. RESULTS: This yeast-based screening system can identify human TOP1 poisons for anticancer efficacy as well as TOP1 suppressors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. CONCLUSIONS: This yeast-based screening system can distinguish between TOP1 suppressors and TOP1 poisons. The assay can also identify compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.

Exonuclease VII repairs quinolone-induced damage by resolving DNA gyrase cleavage complexes.

The widely used quinolone antibiotics act by trapping prokaryotic type IIA topoisomerases, resulting in irreversible topoisomerase cleavage complexes (TOPcc). Whereas the excision repair pathways of TOPcc in eukaryotes have been extensively studied, it is not known whether equivalent repair pathways for prokaryotic TOPcc exist. By combining genetic, biochemical, and molecular biology approaches, we demonstrate that exonuclease VII (ExoVII) excises quinolone-induced trapped DNA gyrase, an essential prokaryotic type IIA topoisomerase. We show that ExoVII repairs trapped type IIA TOPcc and that ExoVII displays tyrosyl nuclease activity for the tyrosyl-DNA linkage on the 5'-DNA overhangs corresponding to trapped type IIA TOPcc. ExoVII-deficient bacteria fail to remove trapped DNA gyrase, consistent with their hypersensitivity to quinolones. We also identify an ExoVII inhibitor that synergizes with the antimicrobial activity of quinolones, including in quinolone-resistant bacterial strains, further demonstrating the functional importance of ExoVII for the repair of type IIA TOPcc.

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies.

The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, ß clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19 000) and CCS range (up to 22 000 Å2 with <0.6% relative standard deviation (RSD)).

Type IA Topoisomerases as Targets for Infectious Disease Treatments.

Infectious diseases are one of the main causes of death all over the world, with antimicrobial resistance presenting a great challenge. New antibiotics need to be developed to provide therapeutic treatment options, requiring novel drug targets to be identified and pursued. DNA topoisomerases control the topology of DNA via DNA cleavage-rejoining coupled to DNA strand passage. The change in DNA topological features must be controlled in vital processes including DNA replication, transcription, and DNA repair. Type IIA topoisomerases are well established targets for antibiotics. In this review, type IA topoisomerases in bacteria are discussed as potential targets for new antibiotics. In certain bacterial pathogens, topoisomerase I is the only type IA topoisomerase present, which makes it a valuable antibiotic target. This review will summarize recent attempts that have been made to identify inhibitors of bacterial topoisomerase I as potential leads for antibiotics and use of these inhibitors as molecular probes in cellular studies. Crystal structures of inhibitor-enzyme complexes and more in-depth knowledge of their mechanisms of actions will help to establish the structure-activity relationship of potential drug leads and develop potent and selective therapeutics that can aid in combating the drug resistant bacterial infections that threaten public health.

DNA and RNA Cleavage Complexes and Repair Pathway for TOP3B RNA- and DNA-Protein Crosslinks.

The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a "self-trapping" mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2.

A diverse global fungal library for drug discovery.

BACKGROUND: Secondary fungal metabolites are important sources for new drugs against infectious diseases and cancers. METHODS: To obtain a library with enough diversity, we collected about 2,395 soil samples and 2,324 plant samples from 36 regions in Africa, Asia, and North America. The collection areas covered various climate zones in the world. We examined the usability of the global fungal extract library (GFEL) against parasitic malaria transmission, Gram-positive and negative bacterial pathogens, and leukemia cells. RESULTS: Nearly ten thousand fungal strains were isolated. Sequences of nuclear ribosomal internal transcribed spacer (ITS) from 40 randomly selected strains showed that over 80% were unique. Screening GFEL, we found that the fungal extract from Penicillium thomii was able to block Plasmodium falciparum transmission to Anopheles gambiae, and the fungal extract from Tolypocladium album was able to kill myelogenous leukemia cell line K562. We also identified a set of candidate fungal extracts against bacterial pathogens.

Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5'-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3'-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains.

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

Covalent Complex of DNA and Bacterial Topoisomerase: Implications in Antibacterial Drug Development.

A topoisomerase-DNA transient covalent complex can be a druggable target for novel topoisomerase poison inhibitors that represent a new class of antibacterial or anticancer drugs. Herein, we have investigated molecular features of the functionally important Escherichia coli topoisomerase I (EctopoI)-DNA covalent complex (EctopoIcc) for molecular simulations, which is very useful in the development of new antibacterial drugs. To demonstrate the usefulness of our approach, we used a model small molecule (SM), NSC76027, obtained from virtual screening. We examined the direct binding of NSC76027 to EctopoI as well as inhibition of EctopoI relaxation activity of this SM via experimental techniques. We then performed molecular dynamics (MD) simulations to investigate the dynamics and stability of EctopoIcc and EctopoI-NSC76027-DNA ternary complex. Our simulation results show that NSC76027 forms a stable ternary complex with EctopoIcc. EctopoI investigated here also serves as a model system for investigating a complex of topoisomerase and DNA in which DNA is covalently attached to the protein.

Inhibition of base excision repair by natamycin suppresses prostate cancer cell proliferation.

Prostate cancer (PCa) progression is characterized by increased expression and transcriptional activity of the androgen receptor (AR). In the advanced stages of prostate cancer, AR significantly upregulates the expression of genes involved in DNA repair. Upregulation of expression for base excision repair (BER) related genes is associated with poor patient survival. Thus, inhibition of the BER pathway may prove to be an effective therapy for prostate cancer. Using a high throughput BER capacity screening assay, we sought to identify BER inhibitors that can synergize with castration therapy. An FDA-approved drug library was screened to identify inhibitors of BER using a fluorescence-based assay suitable for HTS. A gel-based secondary assay confirmed the reduction of BER capacity by compounds identified in the primary screen. Five compounds were then selected for further testing in the independently derived, androgen-dependent prostate cancer cell lines, LNCaP and LAPC4, and in the nonmalignant prostate derived cell lines PNT1A and RWPE1. Further analysis led to the identification of a lead compound, natamycin, as an effective inhibitor of key BER enzymes DNA polymerase ß (pol ß) and DNA Ligase I (LIG I). Natamycin significantly inhibited proliferation of PCa cells in an androgen depleted environment at 1 µM concentration, however, growth inhibition did not occur with nonmalignant prostate cell lines, suggesting that BER inhibition may improve efficacy of the castration therapies.

Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt(III) complex with a thiosemicarbazone ligand.

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)2(O2CO)]Cl·6H2O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)2(O2CO)]Cl·6H2O 2 was used to produce anhydrous [Co(phen)2(H2O)2](NO3)33. Subsequently, anhydrous [Co(phen)2(H2O)2](NO3)33 was reacted with MeATSC 1 to produce [Co(phen)2(MeATSC)](NO3)3·1.5H2O·C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV-visible, and multinuclear NMR (1H, 13C, and 59Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV-visible spectrophotometric studies. UV-visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (Kb = 8.1 × 105 and 1.6 × 104 M-1, respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 ±â€¯5.2 µM when compared to IC50 = 13.75 ±â€¯1.08 µM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨm). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨm, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching.

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes (Escherichia coli and Mycobacterium smegmatis DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes (E. coli DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, K M, V max, and k cat. We found that k cat for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to E. coli DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.

Tyrosyl-DNA Phosphodiesterase 1 and Topoisomerase I Activities as Predictive Indicators for Glioblastoma Susceptibility to Genotoxic Agents.

Glioblastoma (GBM) patients have an estimated survival of ~15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for the selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex (TOP1cc), inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan. We treated GBM cell lines with increasing concentrations of irinotecan and compared the IC50 values. We found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.972, based on the average from three sets of experiments) with IC50 values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by the ectopic expression of wild-type TDP1 increased in irinotecan IC50, while the expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan. The TDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for the selection of individual patients for irinotecan treatment based on risk-benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies.