Publications by authors named "Yuk-Ching Tse-Dinh"

78 Publications

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

Sci Adv 2021 Mar 3;7(10). Epub 2021 Mar 3.

Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA.

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.
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http://dx.doi.org/10.1126/sciadv.abe0384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929499PMC
March 2021

Trapped Ion Mobility Spectrometry of Native Macromolecular Assemblies.

Anal Chem 2021 02 25;93(5):2933-2941. Epub 2021 Jan 25.

Department of Chemistry and Biochemistry, Florida International University, Miami, Florida 33199, United States.

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 ( = 0.185-1.84 cm·V·s), 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 () mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCS) 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 ( = 6-73), Tuning Mix oligomers ( = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin ( = 1-4), carbonic anhydrase, β clamp ( = 1-4), topoisomerase IB, bovine serum albumin ( = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase ( = 1-2)) covering a wide mass (up to / 19 000) and CCS range (up to 22 000 Å with <0.6% relative standard deviation (RSD)).
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http://dx.doi.org/10.1021/acs.analchem.0c04556DOI Listing
February 2021

Type IA Topoisomerases as Targets for Infectious Disease Treatments.

Microorganisms 2021 Jan 1;9(1). Epub 2021 Jan 1.

Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.

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.
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http://dx.doi.org/10.3390/microorganisms9010086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823277PMC
January 2021

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

Cell Rep 2020 Dec;33(13):108569

Developmental Therapeutics Branch & Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2020.108569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859927PMC
December 2020

A diverse global fungal library for drug discovery.

PeerJ 2020 27;8:e10392. Epub 2020 Nov 27.

Department of Biological Sciences, Florida International University, Miami, FL, United States of America.

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 was able to block transmission to , and the fungal extract from was able to kill myelogenous leukemia cell line K562. We also identified a set of candidate fungal extracts against bacterial pathogens.
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http://dx.doi.org/10.7717/peerj.10392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703384PMC
November 2020

Mechanism of Type IA Topoisomerases.

Molecules 2020 Oct 17;25(20). Epub 2020 Oct 17.

Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.

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.
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http://dx.doi.org/10.3390/molecules25204769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587558PMC
October 2020

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

Nucleic Acids Res 2020 05;48(8):4448-4462

Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.

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.
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http://dx.doi.org/10.1093/nar/gkaa201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192597PMC
May 2020

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

ChemMedChem 2020 04 18;15(7):623-631. Epub 2020 Mar 18.

Biomolecular sciences institute, Florida International University, Miami, FL 33199, USA.

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.
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http://dx.doi.org/10.1002/cmdc.201900721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7133791PMC
April 2020

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

Biochimie 2020 Jan 19;168:241-250. Epub 2019 Nov 19.

Department of Human and Molecular Genetics, Florida International University, Herbert Wertheim College of Medicine, Miami, FL, 33199, USA; Department of Chemistry and Biochemistry, Florida International University, Miami, FL, 33199, USA; Biomolecular Sciences Institute, Florida International University, Miami, FL, 33199, USA; Biochemistry Ph.D. Program, Florida International University, Miami, FL, 33199, USA; Department of Cellular and Molecular Biology, Baylor College of Medicine, USA. Electronic address:

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 β (pol β) 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.
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http://dx.doi.org/10.1016/j.biochi.2019.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926147PMC
January 2020

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

ACS Omega 2019 Nov 24;4(19):18413-18422. Epub 2019 Oct 24.

Biomolecular Sciences Institute, Department of Chemistry & Biochemistry, and Enviromental and Occupational Health, Robert Stempel College of Public Health & Social Work, Florida International University, Miami, Florida 33199, United States.

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 ( and DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes ( 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, , , and . We found that 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 DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.
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http://dx.doi.org/10.1021/acsomega.9b02676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844113PMC
November 2019

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

J Inorg Biochem 2020 02 2;203:110907. Epub 2019 Nov 2.

Department of Chemistry and Biochemistry, Old Dominion University, 4541 Hampton Boulevard, Norfolk, VA 23529, USA. Electronic address:

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)(OCO)]Cl·6HO 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)(OCO)]Cl·6HO 2 was used to produce anhydrous [Co(phen)(HO)](NO)3. Subsequently, anhydrous [Co(phen)(HO)](NO)3 was reacted with MeATSC 1 to produce [Co(phen)(MeATSC)](NO)·1.5HO·CHOH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV-visible, and multinuclear NMR (H, C, and Co) 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 (K = 8.1 × 10 and 1.6 × 10 M, 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 (IC = 34.4 ± 5.2 μM when compared to IC = 13.75 ± 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 (ΔΨ). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨ, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.
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http://dx.doi.org/10.1016/j.jinorgbio.2019.110907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053658PMC
February 2020

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

Cancers (Basel) 2019 Sep 23;11(10). Epub 2019 Sep 23.

Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA.

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 IC 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 IC values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by the ectopic expression of wild-type TDP1 increased in irinotecan IC, 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.
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http://dx.doi.org/10.3390/cancers11101416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827102PMC
September 2019

Recent Advances in Use of Topoisomerase Inhibitors in Combination Cancer Therapy.

Curr Top Med Chem 2019 ;19(9):730-740

Department of Chemistry and Biochemistry, Florida International University, Miami, FL, United States.

Inhibitors targeting human topoisomerase I and topoisomerase II alpha have provided a useful chemotherapy option for the treatment of many patients suffering from a variety of cancers. While the treatment can be effective in many patient cases, use of these human topoisomerase inhibitors is limited by side-effects that can be severe. A strategy of employing the topoisomerase inhibitors in combination with other treatments can potentially sensitize the cancer to increase the therapeutic efficacy and reduce resistance or adverse side effects. The combination strategies reviewed here include inhibitors of DNA repair, epigenetic modifications, signaling modulators and immunotherapy. The ongoing investigations on cellular response to topoisomerase inhibitors and newly initiated clinical trials may lead to adoption of novel cancer therapy regimens that can effectively stop the proliferation of cancer cells while limiting the development of resistance.
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http://dx.doi.org/10.2174/1568026619666190401113350DOI Listing
July 2019

Microheterogeneity of Topoisomerase IA/IB and Their DNA-Bound States.

ACS Omega 2019 Feb 18;4(2):3619-3626. Epub 2019 Feb 18.

Department of Chemistry and Biochemistry and Biomolecular Sciences Institute,Florida International University, 11200 SW 8th St., AHC4-233, Miami, Florida 33199, United States.

Topoisomerases are important complex enzymes that modulate DNA topology to maintain chromosome superstructure and integrity. These enzymes are involved in many cellular processes that resolve specific DNA superstructures and intermediates. The low abundance combined with the biological heterogeneity of relevant intermediates of topoisomerases makes their structural information not readily accessible using traditional structural biology tools (e.g., NMR and X-ray crystallography). In the present work, a second-generation trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to study topoisomerase IA (EcTopIA) and virus topoisomerase IB (vTopIB) as well as their complexes with a single-stranded DNA and a stem-loop DNA under native conditions. The higher trapping efficiency and extended mass range of the new, convex TIMS geometry allowed for the separation and identification of multiple conformational states for the two topoisomerases and their DNA complexes. Inspection of the conformational space of EcTopIA and vTopIB in complex with DNA showed that upon DNA binding, the number of conformational states is significantly reduced, suggesting that the DNA binding selects for a narrow range of conformers restricted by the interaction with the DNA substrate. The large microheterogeneity observed for the two DNA binding proteins suggests that they can have multiple biological functions. This work highlights the potential of TIMS-MS for the structural investigations of intrinsically disordered proteins (e.g., DNA binding proteins) as a way to gain a better understanding of the mechanisms involved in DNA substrate recognition, binding, and assembly of the catalytically active enzyme-DNA complex.
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http://dx.doi.org/10.1021/acsomega.8b02887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396120PMC
February 2019

Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound.

PLoS One 2019 22;14(2):e0207733. Epub 2019 Feb 22.

Biomolecular Sciences Institute, Florida International University, Miami, Florida, United States of America.

We have previously reported the inhibition of bacterial topoisomerase I activity by a fluoroquinophenoxazine compound (FP-11g) with a 6-bipiperidinyl lipophilic side chain that exhibited promising antituberculosis activity (MIC = 2.5 μM against Mycobacterium tuberculosis, SI = 9.8). Here, we found that the compound is bactericidal towards Mycobacterium smegmatis, resulting in greater than 5 Log10 reduction in colony-forming units [cfu]/mL following a 10 h incubation at 1.25 μM (4X MIC) concentration. Growth inhibition (MIC = 50 μM) and reduction in cfu could also be observed against a clinical isolate of Mycobacterium abscessus. Stepwise isolation of resistant mutants of M. smegmatis was conducted to explore the mechanism of resistance. Mutations in the resistant isolates were identified by direct comparison of whole-genome sequencing data from mutant and wild-type isolates. These include mutations in genes likely to affect the entry and retention of the compound. FP-11g inhibits Mtb topoisomerase I and Mtb gyrase with IC50 of 0.24 and 27 μM, respectively. Biophysical analysis showed that FP-11g binds DNA as an intercalator but the IC50 for inhibition of Mtb topoisomerase I activity is >10 fold lower than the compound concentrations required for producing negatively supercoiled DNA during ligation of nicked circular DNA. Thus, the DNA-binding property of FP-11g may contribute to its antimycobacterial mechanism, but that alone cannot account for the observed inhibition of Mtb topoisomerase I.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207733PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386362PMC
November 2019

Direct observation of topoisomerase IA gate dynamics.

Nat Struct Mol Biol 2018 12 26;25(12):1111-1118. Epub 2018 Nov 26.

Laboratory of Single Molecule Biophysics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA.
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http://dx.doi.org/10.1038/s41594-018-0158-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379066PMC
December 2018

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

Nucleic Acids Res 2018 08;46(14):7296-7308

Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8 St, Miami, FL 33199, USA.

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.
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http://dx.doi.org/10.1093/nar/gky492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101483PMC
August 2018

Biochemical Basis of Topoisomerase I Relaxation Activity Reduction by Nonenzymatic Lysine Acetylation.

Int J Mol Sci 2018 May 11;19(5). Epub 2018 May 11.

Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA.

The relaxation activity of topoisomerase I is required for regulation of global and local DNA supercoiling. The in vivo topoisomerase I enzyme activity is sensitive to lysine acetylation⁻deacetylation and can affect DNA supercoiling and growth as a result. Nonenzymatic lysine acetylation by acetyl phosphate has been shown to reduce the relaxation activity of topoisomerase I. In this work, the biochemical consequence of topoisomerase I modification by acetyl phosphate with enzymatic assays was studied. Results showed that noncovalent binding to DNA and DNA cleavage by the enzyme were reduced as a result of the acetylation, with greater effect on DNA cleavage. Four lysine acetylation sites were identified using bottom-up proteomics: Lys13, Lys45, Lys346, and Lys488. The Lys13 residue modified by acetyl phosphate has not been reported previously as a lysine acetylation site for topoisomerase I. We discuss the potential biochemical consequence of lysine acetylation at this strictly conserved lysine and other lysine residues on the enzyme based on available genetic and structural information.
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http://dx.doi.org/10.3390/ijms19051439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983628PMC
May 2018

Synthesis of Antimicrobial Poly(guanylurea)s.

Bioconjug Chem 2018 04 12;29(4):1006-1009. Epub 2018 Mar 12.

Department of Chemistry and Biochemistry, Biomolecular Sciences Institute , Florida International University , 11200 SW 8th Street , Miami , Florida 33199 , United States.

Bacterial infections are serious health threats. Emerging drug resistance in bacteria further poses serious challenges to the treatment options involving traditional antibiotics. Antimicrobial polymers disrupt the physical cell membrane integrity of bacteria to address the drug resistance problems. Here, we introduce a conceptually new class of antimicrobial polymers containing positively charged guanylurea backbones for enhanced antimicrobial effects. The initial structure-activity relationship studies demonstrate that poly(guanylurea piperazine)s (PGU-Ps) exhibit excellent antimicrobial activity against different types of bacteria with high selectivity. The new design concept of using a positively charged guanylurea backbone will contribute to the development of future biocompatible, specific, and selective antimicrobial polymers.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00057DOI Listing
April 2018

Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates Targeting Mycobacterium tuberculosis and Mycobacterium abscessus.

Antimicrob Agents Chemother 2018 05 26;62(5). Epub 2018 Apr 26.

Division of Immunity and Pathogenesis, College of Medicine, Biomedical Sciences Department, University of Central Florida, Orlando, Florida, USA

and the fast-growing species are two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistant strains and the high level of intrinsic resistance of call for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), against and We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicating conditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverse and clinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target the gyrase. enzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity against and that act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.
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http://dx.doi.org/10.1128/AAC.01696-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923144PMC
May 2018

Discovery of novel bacterial topoisomerase I inhibitors by use of in silico docking and in vitro assays.

Sci Rep 2018 01 23;8(1):1437. Epub 2018 Jan 23.

Biomolecular Sciences Institute, Florida International University, Miami, FL, 33199, USA.

Topoisomerases are important targets for antibacterial and anticancer therapies. Bacterial topoisomerase I remains to be exploited for antibiotics that can be used in the clinic. Inhibitors of bacterial topoisomerase I may provide leads for novel antibacterial drugs against pathogens resistant to current antibiotics. TB is the leading infectious cause of death worldwide, and new TB drugs against an alternative target are urgently needed to overcome multi-drug resistance. Mycobacterium tuberculosis topoisomerase I (MtbTopI) has been validated genetically and chemically as a TB drug target. Here we conducted in silico screening targeting an active site pocket of MtbTopI. The top hits were assayed for inhibition of MtbTopI activity. The shared structural motif found in the active hits was utilized in a second round of in silico screening and in vitro assays, yielding selective inhibitors of MtbTopI with ICs as low as 2 µM. Growth inhibition of Mycobacterium smegmatis by these compounds in combination with an efflux pump inhibitor was diminished by the overexpression of recombinant MtbTopI. This work demonstrates that in silico screening can be utilized to discover new bacterial topoisomerase I inhibitors, and identifies a novel structural motif which could be explored further for finding selective bacterial topoisomerase I inhibitors.
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http://dx.doi.org/10.1038/s41598-018-19944-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780498PMC
January 2018

A Fluorescence-Based Assay for Identification of Bacterial Topoisomerase I Poisons.

Methods Mol Biol 2018 ;1703:259-268

Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.

Bacterial Topoisomerase I is a potential target for the identification of novel topoisomerase poison inhibitors that could provide leads for a new class of antibacterial compounds. Here we describe in detail a fluorescence-based cleavage assay that is successfully used in HTS for the discovery of bacterial topoisomerase Ι poisons.
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http://dx.doi.org/10.1007/978-1-4939-7459-7_18DOI Listing
July 2018

Biochemical and biophysical properties of positively supercoiled DNA.

Biophys Chem 2017 11 1;230:68-73. Epub 2017 Sep 1.

Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, United States; Department of Chemistry & Biochemistry, Florida International University, Miami, FL 33199, United States. Electronic address:

In this paper we successfully developed a procedure to generate the (+) supercoiled (sc) plasmid DNA template pZXX6 in the milligram range. With the availability of the (+) sc DNA, we are able to characterize and compare certain biochemical and biophysical properties of (+) sc, (-) sc, and relaxed (rx) DNA molecules using different techniques, such as UV melting, circular dichroism, and fluorescence spectrometry. Our results show that (+) sc, (-) sc, and rx DNA templates can only be partially melted due to the fact that these DNA templates are closed circular DNA molecules and the two DNA strands cannot be completely separated upon denaturation at high temperatures. We also find that the fluorescence intensity of a DNA-binding dye SYTO12 upon binding to the (-) sc DNA is significantly higher than that of its binding to the (+) sc DNA. This unique property may be used to differentiate the (-) sc DNA from the (+) sc DNA. Additionally, we demonstrate that E. coli topoisomerase I cannot relax the (+) sc DNA. In contrast, E. coli DNA gyrase can efficiently convert the (+) sc DNA to the (-) sc DNA. Furthermore, our dialysis competition assays show that DNA intercalators prefer binding to the (-) sc DNA.
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http://dx.doi.org/10.1016/j.bpc.2017.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773249PMC
November 2017

Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein-Protein Interaction.

J Mol Biol 2017 09 24;429(19):2931-2942. Epub 2017 Aug 24.

Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA; Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, USA. Electronic address:

We report here a distinct mechanism of interaction between topoisomerase I and RNA polymerase in Mycobacterium tuberculosis and Mycobacterium smegmatis that has evolved independently from the previously characterized interaction between bacterial topoisomerase I and RNA polymerase. Bacterial DNA topoisomerase I is responsible for preventing the hyper-negative supercoiling of genomic DNA. The association of topoisomerase I with RNA polymerase during transcription elongation could efficiently relieve transcription-driven negative supercoiling. Our results demonstrate a direct physical interaction between the C-terminal domains of topoisomerase I (TopoI-CTDs) and the β' subunit of RNA polymerase of M. smegmatis in the absence of DNA. The TopoI-CTDs in mycobacteria are evolutionarily unrelated in amino acid sequence and three-dimensional structure to the TopoI-CTD found in the majority of bacterial species outside Actinobacteria, including Escherichia coli. The functional interaction between topoisomerase I and RNA polymerase has evolved independently in mycobacteria and E. coli, with distinctively different structural elements of TopoI-CTD utilized for this protein-protein interaction. Zinc ribbon motifs in E. coli TopoI-CTD are involved in the interaction with RNA polymerase. For M. smegmatis TopoI-CTD, a 27-amino-acid tail that is rich in basic residues at the C-terminal end is responsible for the interaction with RNA polymerase. Overexpression of recombinant TopoI-CTD in M. smegmatis competed with the endogenous topoisomerase I for protein-protein interactions with RNA polymerase. The TopoI-CTD overexpression resulted in decreased survival following treatment with antibiotics and hydrogen peroxide, supporting the importance of the protein-protein interaction between topoisomerase I and RNA polymerase during stress response of mycobacteria.
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http://dx.doi.org/10.1016/j.jmb.2017.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5610943PMC
September 2017

Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase I by Hoechst 33258 Derived Mono- and Bisbenzimidazoles.

J Med Chem 2017 06 31;60(12):4904-4922. Epub 2017 May 31.

Laboratory of Medicinal Chemistry, Department of Chemistry, Clemson University , Clemson, South Carolina 29634, United States.

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC values <5.0 μM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 μg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 μg/mL, Klebsiella pneumoniae > 32 μg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2-23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00191DOI Listing
June 2017

Deacetylation of topoisomerase I is an important physiological function of E. coli CobB.

Nucleic Acids Res 2017 May;45(9):5349-5358

Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation-deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase.
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http://dx.doi.org/10.1093/nar/gkx250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605244PMC
May 2017

Synthesizing topological structures containing RNA.

Nat Commun 2017 03 31;8:14936. Epub 2017 Mar 31.

Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.

Though knotting and entanglement have been observed in DNA and proteins, their existence in RNA remains an enigma. Synthetic RNA topological structures are significant for understanding the physical and biological properties pertaining to RNA topology, and these properties in turn could facilitate identifying naturally occurring topologically nontrivial RNA molecules. Here we show that topological structures containing single-stranded RNA (ssRNA) free of strong base pairing interactions can be created either by configuring RNA-DNA hybrid four-way junctions or by template-directed synthesis with a single-stranded DNA (ssDNA) topological structure. By using a constructed ssRNA knot as a highly sensitive topological probe, we find that Escherichia coli DNA topoisomerase I has low RNA topoisomerase activity and that the R173A point mutation abolishes the unknotting activity for ssRNA, but not for ssDNA. Furthermore, we discover the topological inhibition of reverse transcription (RT) and obtain different RT-PCR patterns for an ssRNA knot and circle of the same sequence.
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http://dx.doi.org/10.1038/ncomms14936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381007PMC
March 2017

Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking.

FEBS Lett 2017 Jan 20;591(1):28-38. Epub 2016 Dec 20.

Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.

Topoisomerases catalyze changes in DNA topology by directing the movement of DNA strands through consecutive cleavage-rejoining reactions of the DNA backbone. We describe the use of a phenylselenyl-modified thymidine incorporated into a specific position of a partially unwound DNA substrate in crosslinking studies of Escherichia coli topoisomerase I to gain new insights into its catalytic mechanism. Crosslinking of the phenylselenyl-modified thymidine to the topoisomerase protein was achieved by the addition of a mild oxidant. Following nuclease and trypsin digestion, lysine residues on topoisomerase I crosslinked to the modified thymidine were identified by mass spectrometry. The crosslinked sites may correspond to proximal sites for the unwound DNA strand as it interacts with enzyme in the different stages of the catalytic cycle.
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http://dx.doi.org/10.1002/1873-3468.12517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5235945PMC
January 2017

Synthesis, evaluation, and CoMFA study of fluoroquinophenoxazine derivatives as bacterial topoisomerase IA inhibitors.

Eur J Med Chem 2017 Jan 18;125:515-527. Epub 2016 Sep 18.

Department of Pharmaceutical Sciences, The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA. Electronic address:

New antibacterial agents with novel target and mechanism of action are urgently needed to combat problematic bacterial infections and mounting antibiotic resistances. Topoisomerase IA represents an attractive and underexplored antibacterial target, as such, there is a growing interest in developing selective and potent topoisomerase I inhibitors for antibacterial therapy. Based on our initial biological screening, fluoroquinophenoxazine 1 was discovered as a low micromolar inhibitor against E. coli topoisomerase IA. In the literature, fluoroquinophenoxazine analogs have been investigated as antibacterial and anticancer agents, however, their topoisomerase I inhibition was relatively underexplored and there is little structure-activity relationship (SAR) available. The good topoisomerase I inhibitory activity of 1 and the lack of SAR prompted us to design and synthesize a series of fluoroquinophenoxazine analogs to systematically evaluate the SAR and to probe the structural elements of the fluoroquinophenoxazine core toward topoisomerase I enzyme target recognition. In this study, a series of fluoroquinophenoxazine analogs was designed, synthesized, and evaluated as topoisomerase I inhibitors and antibacterial agents. Target-based assays revealed that the fluoroquinophenoxazine derivatives with 9-NH and/or 6-substituted amine functionalities generally exhibited good to excellent inhibitory activities against topoisomerase I with ICs ranging from 0.24 to 3.9 μM. Notably, 11a bearing the 6-methylpiperazinyl and 9-amino motifs was identified as one of the most potent topoisomerase I inhibitors (IC = 0.48 μM), and showed broad spectrum antibacterial activity (MICs = 0.78-7.6 μM) against all the bacteria strains tested. Compound 11g with the 6-bipiperidinyl lipophilic side chain exhibited the most potent antituberculosis activity (MIC = 2.5 μM, SI = 9.8). In addition, CoMFA analysis was performed to investigate the 3D-QSAR of this class of fluoroquinophenoxazine derivatives. The constructed CoMFA model produced reasonable statistics (q = 0.688 and r = 0.806). The predictive power of the developed model was obtained using a test set of 7 compounds, giving a predictive correlation coefficient r of 0.767. Collectively, these promising data demonstrated that fluoroquinophenoxazine derivatives have the potential to be developed as a new chemotype of potent topoisomerase IA inhibitors with antibacterial therapeutic potential.
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http://dx.doi.org/10.1016/j.ejmech.2016.09.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148682PMC
January 2017

Characterization of molecular interactions between Escherichia coli RNA polymerase and topoisomerase I by molecular simulations.

FEBS Lett 2016 09 4;590(17):2844-51. Epub 2016 Aug 4.

Biomolecular Sciences Institute, Florida International University, Miami, FL, USA.

Escherichia coli topoisomerase I (EctopoI), a type IA DNA topoisomerase, relaxes the negative DNA supercoiling generated by RNA polymerase (RNAP) during transcription elongation. Due to the lack of structural information on the complex, the exact nature of the RNAP-EctopoI interactions remains unresolved. Herein, we report for the first time, the structure-based modeling of the RNAP-EctopoI interactions using computational methods. Our results predict that the salt bridge as well as hydrogen bond interactions are responsible for the formation and stabilization of the RNAP-EctopoI complex. Our investigations provide molecular insights for understanding how EctopoI interacts with RNAP, a critical step for preventing hypernegative DNA supercoiling during transcription.
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http://dx.doi.org/10.1002/1873-3468.12321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014613PMC
September 2016