Secondary literature sources for TOPEUc
The following references were automatically generated.
- Rajan R, Osterman AK, Gast AT, Mondragon A
- Biochemical characterization of the topoisomerase domain of Methanopyrus kandleri topoisomerase V.
- J Biol Chem. 2014; 289: 28898-909
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Topoisomerases are ubiquitous enzymes that modify the topological state of DNA inside the cell and are essential for several cellular processes. Topoisomerase V is the sole member of the type IC topoisomerase subtype. The topoisomerase domain has a unique fold among topoisomerases, and the putative active site residues show a distinct arrangement. The present study was aimed at identifying the roles of the putative active site residues in the DNA cleavage/religation process. Residues Arg-131, Arg-144, His-200, Glu-215, Lys-218, and Tyr-226 were mutated individually to a series of conservative and non-conservative amino acids, and the DNA relaxation activity at different pH values, times, and enzyme concentrations was compared with wild-type activity. The results suggest that Arg-144 is essential for protein stability because any substitution at this position was deleterious and that Arg-131 and His-200 are involved in transition state stabilization. Glu-215 reduces the DNA binding ability of topoisomerase V, especially in shorter fragments with fewer helix-hairpin-helix DNA binding motifs. Finally, Lys-218 appears to play a direct role in catalysis but not in charge stabilization of the protein-DNA intermediate complex. The results suggest that although catalytically important residues are oriented in different fashions in the active sites of type IB and type IC topoisomerases, similar amino acids play equivalent roles in both of these subtypes of enzymes, showing convergent evolution of the catalytic mechanism.
- Shin DS, Pratt AJ, Tainer JA
- Archaeal genome guardians give insights into eukaryotic DNA replication and damage response proteins.
- Archaea. 2014; 2014: 206735-206735
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As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine.
- Siu FM, Pommier Y
- Sequence selectivity of the cleavage sites induced by topoisomerase I inhibitors: a molecular dynamics study.
- Nucleic Acids Res. 2013; 41: 10010-9
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Topoisomerase IB (Top1) inhibitors, such as camptothecin (CPT), stabilize the Top1-DNA cleavage complex in a DNA sequence-dependent manner. The sequence selectivity of Top1 inhibitors is important for targeting specific genomic sequences of therapeutic value. However, the molecular mechanisms underlying this selectivity remain largely unknown. We performed molecular dynamics simulations to delineate structural, dynamic and energetic features that contribute to the differential sequence selectivity of the Top1 inhibitors. We found the sequence selectivity of CPT to be highly correlated with the drug binding energies, dynamic and structural properties of the linker domain. Chemical insights, gained by per-residue binding energy analysis revealed that the non-polar interaction between CPT and nucleotide at the +1 position of the cleavage site was the major (favorable) contributor to the total binding energy. Mechanistic insights gained by a potential of mean force analysis implicated that the drug dissociation step was associated with the sequence selectivity. Pharmaceutical insights gained by our molecular dynamics analyses explained why LMP-776, an indenoisoquinoline derivative under clinical development at the National Institutes of Health, displays different sequence selectivity when compared with camptothecin and its clinical derivatives.
- Saikia N, Deka RC
- Ab initio study on the noncovalent adsorption of camptothecin anticancer drug onto graphene, defect modified graphene and graphene oxide.
- J Comput Aided Mol Des. 2013; 27: 807-21
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The application of graphene and related nanomaterials like boron nitride (BN) nanosheets, BN-graphene hybrid nanomaterials, and graphene oxide (GO) for adsorption of anticancer chemotherapeutic camptothecin (CPT) along with the effect on electronic properties prior to functionalization and after functionalization has been reported using density functional theory (DFT) calculations. The inclusion of dispersion correction to DFT is instrumental in accounting for van der Waals pi-pi stacking between CPT and the nanomaterial. The adsorption of CPT exhibits significant strain within the nanosheets and noncovalent adsorption of CPT is thermodynamically favoured onto the nanosheets. In case of GO, surface incorporation of functional groups result in significant crumpling along the basal plane and the interaction is basically mediated by H-bonding rather than pi-pi stacking. Docking studies predict the plausible binding of CPT, CPT functionalized graphene and GO with topoisomerase I (top 1) signifying that CPT interacts through pi stacking with AT and GC base pairs of DNA and in presence of nano support, DNA bases preferentially gets bound to the basal plane of graphene and GO rather than the edges. At a theoretical level of understanding, our studies point out the noncovalent interaction of CPT with graphene based nanomaterials and GO for loading and delivery of anticancer chemotherapeutic along with active binding to Top1 protein.
- Ivanova B, Spiteller M
- Structure and properties of camptothecin derivatives, their protonated forms, and model interaction with the topoisomerase I-DNA complex.
- Biopolymers. 2012; 97: 134-44
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The structure and properties of the 11 Camptothecin derivatives (CPTs) and their different mono-, di-, and triprotonated forms, depending on the number of proton accepting centers in the molecules are studied both theoretically and experimentally by quantum chemical approaches, electronic absorption, and CD spectroscopy. The study of the protonated forms of the CPTs and search of the electron-withdrawing groups is crucial of the water-solubility of the novel medications. Thus, the model interaction of the different protonated molecular species with the Topoisomerase I-DNA complex are elucidated and discussed with a view to understand the mode of binding of the CPTs depending on the type of the substituents and pH of the medium.
- Zare-Mirakabadi A, Sarzaeem A, Moradhaseli S, Sayad A, Negahdary M
- Necrotic Effect versus Apoptotic Nature of Camptothecin in Human Cervical Cancer Cells.
- Iran J Cancer Prev. 2012; 5: 109-16
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BACKGROUND: Functional defects in mitochondria are involved in the induction of cell death in cancer cells. The process of programmed cell death may occur through the mechanisms of apoptosis. Several potential lead molecules such as Camptothecin (CPT) and its analogues have been isolated from plants with anticancer effect. The aim of the present study was to understand the necrotic effect versus apoptotic nature of CPT in HeLa cancer cells. METHODS: The anti-proliferative activity of CPT was estimated through 3-(4, 5- Dimethyl Thiazol-2-yl)-2, 5-diphenyl Tetrazolium bromide (MTT) assay and DNA fragmentation analysis using gel electrophoresis. Lactate Dehydrogenase (LDH) activity and cell morphology were assessed under control and CPT exposed conditions to evaluate the necrotic effect of CPT. RESULTS: The results showed that CPT inhibited the proliferation of HeLa cells in a dose-dependent manner with an Inhibitory Concentration 50% (IC50) of 0.08+/-0.012 microg/ml. However the significant (p<0.05) increase happens in LDH activity at concentrations 1x10(-1)microg/ml and above. Morphological changes showed that CPT in low concentrations induced an apoptotic mechanism of cell death, such as cell shrinkage and characteristic rounding of dying cells, while at high concentrations showed necrosis changes. The characteristic DNA ladder formation of CPT-treated cells in agarose gel electrophoresis confirmed the results obtained by light microscopy and LDH assay. CONCLUSION: Camptothecin as an anticancer drug may have anti-proliferative effect on HeLa cancer cells in low concentrations, through its nature of induction of apoptosis. The border line between necrotic effect and apoptotic nature of CPT in HeLa cancer cells has been found to be at concentration of 1x10(-1) microg/ml.
- Kiselev E et al.
- 7-azaindenoisoquinolines as topoisomerase I inhibitors and potential anticancer agents.
- J Med Chem. 2011; 54: 6106-16
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A series of 7-azaindenoisoquinoline topoisomerase I (Top1) inhibitors have been prepared to investigate the effect of increased electron affinity of the aromatic system on the ability to stabilize the Top1-DNA cleavage complex. Ab initio calculations suggest that introduction of nitrogen into the aromatic system of the indenoisoquinolines would facilitate charge transfer complex formation with DNA, thus improving the pi-pi stacking interactions. The present study shows that 7-azaindenoisoquinolines demonstrate improved water solubility without any decrease in Top1 inhibitory activity or cytotoxicity. Analysis of the biological results reveals that smaller lactam ring substituents enable intercalation into both free DNA and Top1-DNA cleavage complex, whereas larger substituents only allow binding to the cleavage complex but not free DNA. Free DNA binding suppresses Top1-catalyzed DNA cleavage at high drug concentrations, whereas DNA cleavage and inhibition of religation occurs at low drug concentration.
- Fukui T et al.
- Prevalence of topoisomerase I genetic mutations and UGT1A1 polymorphisms associated with irinotecan in individuals of Asian descent.
- Oncol Lett. 2011; 2: 923-928
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Topoisomerase I (TOP-I) mutations have been shown to be correlated to irinotecan resistance in vitro. However, the prevalence of TOP-I germline mutations has yet to be systematically elucidated. On the other hand, polymorphisms of UGT1A1 have been shown to be associated with CPT-11 toxicity in clinical situations. The primary aim of this study was to investigate the prevalence of mutations in the TOP-I exons associated with CPT-11 resistance, including untreated cancer tissue. A secondary aim was to confirm the less frequent UGT1A1*28 and more frequent UGT1A1*6 in individuals of Asian descent compared to Caucasians and individuals of African descent. The prevalence of 5 reported TOP-I mutations in exons was investigated in volunteers (n=236) using DNA sequencing of the PCR products. The prevalence of TOP-I mutations in untreated lung cancer tissues (n=16) was also investigated. Additionally, 3 UGT1A1 polymorphisms, UGT1A1*6, *27 and *28, were investigated in volunteers (n=126). There were no mutations of TOP-I in any of the 236 subjects or in the untreated lung tissues. Among 128 subjects, the distribution of homozygous polymorphisms of UGT1A1 was: UGT1A1*28 in 3 (2.4%) and UGT1A1*6 in 4 (3.2%) subjects, and co-occurrence of heterozygous polymorphisms for both UGT1A1*6 and UGT1A1*28 in 4 (3.2%) subjects, and for UGT1A1*27 and UGT1A1*28 in 1 subject (0.8%). The Hardy-Weinberg deviation test showed there was no significant deviation from the equilibrium, and the association analysis indicated no significant linkage between UGT1A1*6 and UGT1A1*28. In conclusion, TOP-I genetic mutations correlated to CPT-11 resistance were not detected in any of the subjects and untreated lung cancer tissues. Less frequent UGT1A1*28 and more frequent UGT1A1*6 were confirmed in East Asian individuals compared to Caucasians and individuals of African descent. Linkage disequilibrium was not detected between UGT1A1*6 and UGT1A1*28.
- Vos SM, Tretter EM, Schmidt BH, Berger JM
- All tangled up: how cells direct, manage and exploit topoisomerase function.
- Nat Rev Mol Cell Biol. 2011; 12: 827-41
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Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.
- Koster DA, Crut A, Shuman S, Bjornsti MA, Dekker NH
- Cellular strategies for regulating DNA supercoiling: a single-molecule perspective.
- Cell. 2010; 142: 519-30
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Entangling and twisting of cellular DNA (i.e., supercoiling) are problems inherent to the helical structure of double-stranded DNA. Supercoiling affects transcription, DNA replication, and chromosomal segregation. Consequently the cell must fine-tune supercoiling to optimize these key processes. Here, we summarize how supercoiling is generated and review experimental and theoretical insights into supercoil relaxation. We distinguish between the passive dissipation of supercoils by diffusion and the active removal of supercoils by topoisomerase enzymes. We also review single-molecule studies that elucidate the timescales and mechanisms of supercoil removal.
- Kiselev E, Dexheimer TS, Pommier Y, Cushman M
- Design, synthesis, and evaluation of dibenzo[c,h][1,6]naphthyridines as topoisomerase I inhibitors and potential anticancer agents.
- J Med Chem. 2010; 53: 8716-26
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Indenoisoquinoline topoisomerase I (Top1) inhibitors are a novel class of anticancer agents. Modifications of the indenoisoquinoline A, B, and D rings have been extensively studied in order to optimize Top1 inhibitory activity and cytotoxicity. To improve understanding of the forces that stabilize drug-Top1-DNA ternary complexes, the five-membered cyclopentadienone C-ring of the indenoisoquinoline system was replaced by six-membered nitrogen heterocyclic rings, resulting in dibenzo[c,h][1,6]naphthyridines that were synthesized by a novel route and tested for Top1 inhibition. This resulted in several compounds that have unique DNA cleavage site selectivities and potent antitumor activities in a number of cancer cell lines.
- Laponogov I, Pan XS, Veselkov DA, McAuley KE, Fisher LM, Sanderson MR
- Structural basis of gate-DNA breakage and resealing by type II topoisomerases.
- PLoS One. 2010; 5: 11338-11338
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Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg(2+) ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics.
- Patel A, Yakovleva L, Shuman S, Mondragon A
- Crystal structure of a bacterial topoisomerase IB in complex with DNA reveals a secondary DNA binding site.
- Structure. 2010; 18: 725-33
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Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a approximately 21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.
- Baker NM, Rajan R, Mondragon A
- Structural studies of type I topoisomerases.
- Nucleic Acids Res. 2009; 37: 693-701
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Topoisomerases are ubiquitous proteins found in all three domains of life. They change the topology of DNA via transient breaks on either one or two of the DNA strands to allow passage of another single or double DNA strand through the break. Topoisomerases are classified into two types: type I enzymes cleave one DNA strand and pass either one or two DNA strands through the break before resealing it, while type II molecules cleave both DNA strands in concert and pass another double strand through the break followed by religation of the double strand break. Here we review recent work on the structure of type I enzymes. These structural studies are providing atomic details that, together with the existing wealth of biochemical and biophysical data, are bringing our understanding of the mechanism of action of these enzymes to the atomic level.
- Malina J, Vrana O, Brabec V
- Mechanistic studies of the modulation of cleavage activity of topoisomerase I by DNA adducts of mono- and bi-functional PtII complexes.
- Nucleic Acids Res. 2009; 37: 5432-42
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Using electrophoresis and replication mapping, we show that the presence of DNA adducts of bifunctional antitumor cisplatin or monodentate [PtCl(dien)]Cl (dien = diethylenetriamine) in the substrate DNA inhibits eukaryotic topoisomerase 1 (top1) action, the adducts of cisplatin being more effective. The presence of camptothecin in the samples of platinated DNA markedly enhances effects of Pt-DNA adducts on top1 activity. Interestingly, the effects of Pt-DNA adducts on the catalytic activity of top1 in the presence of camptothecin differ depending on the sequence context. A multiple metallation of the short nucleotide sequences on the scissile strand, immediately downstream of the cleavage site impedes the cleavage by top1. On the other hand, DNA cleavage by top1 at some cleavage sites which were not platinated in their close proximity is notably enhanced as a consequence of global platination of DNA. We suggest that this enhancement of DNA cleavage by top1 may consist in its inability to bind to other cleavage sites platinated in their close neighborhood; thus, more molecules of top1 may become available for cleavage at the sites where top1 normally cleaves and where platination does not interfere.
- Schoeffler AJ, Berger JM
- DNA topoisomerases: harnessing and constraining energy to govern chromosome topology.
- Q Rev Biophys. 2008; 41: 41-101
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DNA topoisomerases are a diverse set of essential enzymes responsible for maintaining chromosomes in an appropriate topological state. Although they vary considerably in structure and mechanism, the partnership between topoisomerases and DNA has engendered commonalities in how these enzymes engage nucleic acid substrates and control DNA strand manipulations. All topoisomerases can harness the free energy stored in supercoiled DNA to drive their reactions; some further use the energy of ATP to alter the topology of DNA away from an enzyme-free equilibrium ground state. In the cell, topoisomerases regulate DNA supercoiling and unlink tangled nucleic acid strands to actively maintain chromosomes in a topological state commensurate with particular replicative and transcriptional needs. To carry out these reactions, topoisomerases rely on dynamic macromolecular contacts that alternate between associated and dissociated states throughout the catalytic cycle. In this review, we describe how structural and biochemical studies have furthered our understanding of DNA topoisomerases, with an emphasis on how these complex molecular machines use interfacial interactions to harness and constrain the energy required to manage DNA topology.
- Bouthier de la Tour C, Amrani L, Cossard R, Neuman KC, Serre MC, Duguet M
- Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase.
- J Biol Chem. 2008; 283: 27395-402
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Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.
- Girstun A, Kowalska-Loth B, Czubaty A, Klocek M, Staron K
- Fragment responsible for translocation in the N-terminal domain of human topoisomerase I.
- Biochem Biophys Res Commun. 2008; 366: 250-7
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The N-terminal domain is a fragment that binds proteins and anchors topoisomerase I in the nucleolus. As a separate polypeptide, it translocates from the nucleolus to nucleoplasm upon camptothecin treatment. In this paper, we show that the translocation depends on the short fragment of the domain (residues from 1 to 67). We also present a list of proteins that specifically bind to the fragment responsible for translocation.
- Crut A, Nair PA, Koster DA, Shuman S, Dekker NH
- Dynamics of phosphodiester synthesis by DNA ligase.
- Proc Natl Acad Sci U S A. 2008; 105: 6894-9
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Ligases are essential actors in DNA replication, recombination, and repair by virtue of their ability to seal breaks in the phosphodiester backbone. Ligation proceeds through a nicked DNA-adenylate intermediate (AppDNA), which must be sealed quickly to avoid creating a potentially toxic lesion. Here, we take advantage of ligase-catalyzed AMP-dependent incision of a single supercoiled DNA molecule to observe the step of phosphodiester synthesis in real time. An exponentially distributed number of supercoils was relaxed per successful incision-resealing event, from which we deduce the torque-dependent ligation probability per DNA swivel. Premature dissociation of ligase from nicked DNA-adenylate accounted for approximately 10% of the observed events. The ability of ligase to form a C-shaped protein clamp around DNA is a key determinant of ligation probability per turn and the stability of the ligase-AppDNA intermediate. The estimated rate of phosphodiester synthesis by DNA ligase (400 s(-1)) is similar to the high rates of phosphodiester synthesis by replicative DNA polymerases.
- Darcy IK, Scharein RG, Stasiak A
- 3D visualization software to analyze topological outcomes of topoisomerase reactions.
- Nucleic Acids Res. 2008; 36: 3515-21
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The action of various DNA topoisomerases frequently results in characteristic changes in DNA topology. Important information for understanding mechanistic details of action of these topoisomerases can be provided by investigating the knot types resulting from topoisomerase action on circular DNA forming a particular knot type. Depending on the topological bias of a given topoisomerase reaction, one observes different subsets of knotted products. To establish the character of topological bias, one needs to be aware of all possible topological outcomes of intersegmental passages occurring within a given knot type. However, it is not trivial to systematically enumerate topological outcomes of strand passage from a given knot type. We present here a 3D visualization software (TopoICE-X in KnotPlot) that incorporates topological analysis methods in order to visualize, for example, knots that can be obtained from a given knot by one intersegmental passage. The software has several other options for the topological analysis of mechanisms of action of various topoisomerases.
- Liu P, Ewis HE, Tai PC, Lu CD, Weber IT
- Crystal structure of the Geobacillus stearothermophilus carboxylesterase Est55 and its activation of prodrug CPT-11.
- J Mol Biol. 2007; 367: 212-23
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Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.
- Chillemi G, Bruselles A, Fiorani P, Bueno S, Desideri A
- The open state of human topoisomerase I as probed by molecular dynamics simulation.
- Nucleic Acids Res. 2007; 35: 3032-8
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The open state of human topoisomerase I has been probed by molecular dynamics simulation, starting from the coordinates of the closed structure of the protein complexed with DNA, after elimination of the 22-bp DNA duplex oligonucleotide. A repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum, after which an unperturbed simulation has been carried out for 10 ns. The simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains, which however move as a coherent unit, fully maintaining their secondary and tertiary structures. Despite movements between the domains as large as 80-90 A, the catalytic pentad remains preassembled, the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7 A. Electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center, in a conformation perfectly suited to interact with the negatively charged supercoiled DNA substrate.
- Stivers JT, Nagarajan R
- Probing enzyme phosphoester interactions by combining mutagenesis and chemical modification of phosphate ester oxygens.
- Chem Rev. 2006; 106: 3443-67
- Banerjee A, Santos WL, Verdine GL
- Structure of a DNA glycosylase searching for lesions.
- Science. 2006; 311: 1153-7
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DNA glycosylases must interrogate millions of base pairs of undamaged DNA in order to locate and then excise one damaged nucleobase. The nature of this search process remains poorly understood. Here we report the use of disulfide cross-linking (DXL) technology to obtain structures of a bacterial DNA glycosylase, MutM, interrogating undamaged DNA. These structures, solved to 2.0 angstrom resolution, reveal the nature of the search process: The protein inserts a probe residue into the helical stack and severely buckles the target base pair, which remains intrahelical. MutM therefore actively interrogates the intact DNA helix while searching for damage.
- Huang YY et al.
- The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA).
- Nucleic Acids Res. 2006; 34: 5650-9
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As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.
- Leppard JB, Champoux JJ
- Human DNA topoisomerase I: relaxation, roles, and damage control.
- Chromosoma. 2005; 114: 75-85
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Human DNA topoisomerase I is an essential enzyme involved in resolving the torsional stress associated with DNA replication, transcription, and chromatin condensation. The catalytic cycle of the enzyme consists of DNA cleavage to form a covalent enzyme-DNA intermediate, DNA relaxation, and finally, re-ligation of the phosphate backbone to restore the continuity of the DNA. Structure/function studies have elucidated a flexible enzyme that relaxes DNA through coordinated, controlled movements of distinct enzyme domains. The cellular roles of topoisomerase I are apparent throughout the nucleus, but the concentration of processes acting on ribosomal DNA results in topoisomerase I accumulation in the nucleolus. Although the activity of topoisomerase I is required in these processes, the enzyme can also have a deleterious effect on cells. In the event that the final re-ligation step of the reaction cycle is prevented, the covalent topoisomerase I-DNA intermediate becomes a toxic DNA lesion that must be repaired. The complexities of the relaxation reaction, the cellular roles, and the pathways that must exist to repair topoisomerase I-mediated DNA damage highlight the importance of continued study of this essential enzyme.
- Alessandri M, Beretta GL, Ferretti E, Mancia A, Khobta A, Capranico G
- Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins.
- J Mol Biol. 2004; 337: 295-305
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Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.
- Viard T, Cossard R, Duguet M, de La Tour CB
- Thermotoga maritima-Escherichia coli chimeric topoisomerases. Answers about involvement of the carboxyl-terminal domain in DNA topoisomerase I-mediated catalysis.
- J Biol Chem. 2004; 279: 30073-80
- Display abstract
Bacterial topoisomerases I are generally composed of two domains as follows: a core domain, which contains all the conserved motifs involved in the trans-esterification reactions, and a carboxyl-terminal domain, highly variable in size and sequence. In the present work, we have addressed the question of the respective roles of the two domains in the different steps of the topoisomerization cycle. For this purpose, we prepared various recombinant topoisomerases from two model enzymes: topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima and topoisomerase I from Escherichia coli. We compared the properties of the two core domains to that of the topoisomerases formed by combining the core domain of one enzyme to the carboxyl-terminal domain of the other. We found that, contrary to E. coli (Lima, C. D., Wang, J. C., and Mondragon, A. (1993) J. Mol. Biol. 232, 1213-1216), the core domain from T. maritima (TmTop65) is able to sustain by itself a complete topoisomerization cycle, although with low efficiency. Fusion of TmTop65 to the entire carboxyl-terminal domain from E. coli considerably increases binding efficiency, thermal stability, and DNA relaxation activity. Moreover, the chimera predominantly acquires the cleavage specificity of E. coli full-length topoisomerase. For the chimera obtained by fusion of the T. maritima carboxyl-terminal domain to the core EcTop67, very low DNA relaxation activity and binding are recovered, but formation of a covalent DNA adduct is impaired. Taken together, our results show that the presence and the nature of the carboxyl-terminal domain of bacterial topoisomerases I strongly determine their DNA binding efficiency and cleavage specificity but is not strictly required for strand passage.
- Sauve S, Tremblay L, Lavigne P
- The NMR solution structure of a mutant of the Max b/HLH/LZ free of DNA: insights into the specific and reversible DNA binding mechanism of dimeric transcription factors.
- J Mol Biol. 2004; 342: 813-32
- Display abstract
Basic region-helix1-loop-helix2-leucine zipper (b/H(1)LH(2)/LZ) transcription factors bind specific DNA sequence in their target gene promoters as dimers. Max, a b/H(1)LH(2)/LZ transcription factor, is the obligate heterodimeric partner of the related b/H(1)LH(2)/LZ proteins of the Myc and Mad families. These heterodimers specifically bind E-box DNA sequence (CACGTG) to activate (e.g. c-Myc/Max) and repress (e.g. Mad1/Max) transcription. Max can also homodimerize and bind E-box sequences in c-Myc target gene promoters. While the X-ray structure of the Max b/H(1)LH(2)/LZ/DNA complex and that of others have been reported, the precise sequence of events leading to the reversible and specific binding of these important transcription factors is still largely unknown. In order to provide insights into the DNA binding mechanism, we have solved the NMR solution structure of a covalently homodimerized version of a Max b/H(1)LH(2)/LZ protein with two stabilizing mutations in the LZ, and characterized its backbone dynamics from (15)N spin-relaxation measurements in the absence of DNA. Apart from minor differences in the pitch of the LZ, possibly resulting from the mutations in the construct, we observe that the packing of the helices in the H(1)LH(2) domain is almost identical to that of the two crystal structures, indicating that no important conformational change in these helices occurs upon DNA binding. Conversely to the crystal structures of the DNA complexes, the first 14 residues of the basic region are found to be mostly unfolded while the loop is observed to be flexible. This indicates that these domains undergo conformational changes upon DNA binding. On the other hand, we find the last four residues of the basic region form a persistent helical turn contiguous to H(1). In addition, we provide evidence of the existence of internal motions in the backbone of H(1) that are of larger amplitude and longer time-scale (nanoseconds) than the ones in the H(2) and LZ domain. Most interestingly, we note that conformers in the ensemble of calculated structures have highly conserved basic residues (located in the persistent helical turn of the basic region and in the loop) known to be important for specific binding in a conformation that matches that of the DNA-bound state. These partially prefolded conformers can directly fit into the major groove of DNA and as such are proposed to lie on the pathway leading to the reversible and specific DNA binding. In these conformers, the conserved basic side-chains form a cluster that elevates the local electrostatic potential and could provide the necessary driving force for the generation of the internal motions localized in the H(1) and therefore link structural determinants with the DNA binding function. Overall, our results suggests that the Max homodimeric b/H(1)LH(2)/LZ can rapidly and preferentially bind DNA sequence through transient and partially prefolded states and subsequently, adopt the fully helical bound state in a DNA-assisted mechanism or induced-fit.
- Bugreev DV, Buneva VN, Nevinskii GA
- [The mechanism of specific cleavage of supercoiled DNA by human DNA topoisomerase I: the effect of ligand structure on the catalytic step of reaction].
- Mol Biol (Mosk). 2003; 37: 325-39
- Display abstract
Eukaryotic DNA topoisomerase I (Topo) regulates the topological state of cell DNA and plays an important part in replication, transcription, repair, and recombination. Factors affecting the specific recognition of topologically stressed DNA were analyzed on the basis of the thermodynamic and kinetic data on the Topo-DNA interaction and the X-ray data on human Topo. A model was advanced for possible structural changes occurring in the ligand after initial recognition. The effect of conformational changes in specific DNA on the catalytic stage of the reaction was analyzed.
- Christensen MO, Barthelmes HU, Boege F, Mielke C
- Residues 190-210 of human topoisomerase I are required for enzyme activity in vivo but not in vitro.
- Nucleic Acids Res. 2003; 31: 7255-63
- Display abstract
DNA-topoisomerase I (topo I) unwinds the DNA- double helix by cutting one strand and allowing rotation of the other. In vitro, this function does not require the N-terminal domain of the enzyme, which is believed to regulate cellular properties. To assess this role, we studied the cellular distribution and mobility of green fluorescent protein-chimera of human topo I lacking either the entire N-terminal domain or a portion of it. We find that topo I truncated up to position 210 is not stabilized by camptothecin in covalent DNA-complexes inside a living cell, whereas in vitro it retains full DNA-relaxation activity, and is targeted by camptothecin in the usual manner. This difference is not shared with a fragment lacking the N-terminal domain up to position 190, indicating that residues 190-210 play a crucial role for the activity of the enzyme in its physiological environment, but not in vitro. Since it is impossible to discriminate in vivo whether this region is required for topo I to form covalent DNA intermediates in the cell, or just for camptothecin to bind and stabilize such complexes, we could not explain precisely these cellular observations. However, inactivity in vivo of the enzyme lacking this region is indicated by a lesser cytotoxicity.
- Fiorani P, Bruselles A, Falconi M, Chillemi G, Desideri A, Benedetti P
- Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I.
- J Biol Chem. 2003; 278: 43268-75
- Display abstract
DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.
- Matoba K, Mayanagi K, Nakasu S, Kikuchi A, Morikawa K
- Three-dimensional electron microscopy of the reverse gyrase from Sulfolobus tokodaii.
- Biochem Biophys Res Commun. 2002; 297: 749-55
- Display abstract
Reverse gyrase is a type IA topoisomerase, found in various hyperthermophiles and promotes ATP-dependent positive supercoiling of DNA. Electron microscopy combined with single particle analyses revealed the three-dimensional structure of the DNA-free Sulfolobus tokodaii reverse gyrase and two-dimensional average images of both the protein alone and that complexed with double-stranded DNA. The 23A resolution map exhibited a parallelogrammatic morphology of 110 x 87 x 43A, which is in good agreement with the crystal structure of the Archaeoglobus fulgidus reverse gyrase. The average image of the complex revealed that the monomeric enzyme binds DNA duplex. Together with this average image of the complex, the three-dimensional map implies that, at the beginning of the supercoiling reaction, DNA is bound within a 10-20A wide cleft in the helicase-like domain. We also speculate that DNA may pass through a 20A wide hole at the end of the cleft.
- Trakselis MA, Alley SC, Abel-Santos E, Benkovic SJ
- Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer.
- Proc Natl Acad Sci U S A. 2001; 98: 8368-75
- Display abstract
The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.
- Pouliot JJ, Robertson CA, Nash HA
- Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae.
- Genes Cells. 2001; 6: 677-87
- Display abstract
BACKGROUND: The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double-strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished. RESULTS: Reaction kinetics with model substrates reveal that the catalytic efficiency of Saccharomyces cerevisiae Tdp1 is relatively poor when the scissile bond is located in the middle of a duplex, but much better when it is located at the end of a structure. Survival of yeast after induction of a toxic topoisomerase is substantially reduced by inactivation of the TDP1 gene. Comparison of survival of single and double mutants places TDP1 and RAD52 in the same epistasis group but TDP1 and RAD9 in different epistasis groups. In the absence of RAD9, inactivation of TDP1 has a significant effect on the survival of cells following exposure to camptothecin but is without consequence for the survival of agents that do not target topoisomerase I. CONCLUSIONS: Tdp1 acts as a specific repair enzyme for topoisomerase I lesions. Rather than working at their earliest occurrence, the enzyme acts after covalent complexes have been converted to DSBs. A second repair pathway also exists that functions independently of Tdp1 but requires RAD9 function to efficiently repair topoisomerase I-linked DSBs. The efficiency of these pathways differs for complexes induced with the chemotherapeutic agent camptothecin vs. those accumulated by mutant forms of topoisomerase I.
- Bailly C
- DNA relaxation and cleavage assays to study topoisomerase I inhibitors.
- Methods Enzymol. 2001; 340: 610-23
- Urasaki Y et al.
- Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.
- Cancer Res. 2001; 61: 1964-9
- Display abstract
In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.
- Li Z, Mondragon A, DiGate RJ
- The mechanism of type IA topoisomerase-mediated DNA topological transformations.
- Mol Cell. 2001; 7: 301-7
- Display abstract
Type IA DNA topoisomerases possess several domains forming a toroidal molecule with a central hole large enough to accommodate single- or double-stranded DNA. The sign inversion model predicts several protein-DNA intermediates, including those in which DNA is trapped within the hole. Opposing cysteine residues were incorporated into two independent domains surrounding the putative DNA binding cavity of E. coli topoisomerase III, creating a molecule that can be covalently closed or opened by oxidizing or reducing the disulfide bond. The formation of the disulfide bond allowed the trapping of single- and double-stranded DNA within the cavity of the enzyme and the identification of other intermediates proposed by the sign inversion model.
- Morikawa K, Shirakawa M
- Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA.
- Mutat Res. 2000; 460: 257-75
- Display abstract
Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.
- Syrovets T, Buchele B, Gedig E, Slupsky JR, Simmet T
- Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and IIalpha.
- Mol Pharmacol. 2000; 58: 71-81
- Display abstract
Acetyl-boswellic acids (acetyl-BA) are pentacyclic triterpenes derived from the gum resin of frankincense. We have previously shown that these compounds are effective cytotoxic agents, acting through a mechanism that appears to involve the inhibition of topoisomerase activity. We have now investigated the mechanism of action of acetyl-BA and show that these compounds are more potent inhibitors of human topoisomerases I and IIalpha than camptothecin, and amsacrine or etoposide, respectively. Our data demonstrate that acetyl-BA and, to a lesser extent, some other pentacyclic triterpenes, such as betulinic acid, ursolic acid, and oleanolic acid, inhibit topoisomerases I and IIalpha through a mechanism that does not involve stabilization of the cleavable complex or the intercalation of DNA. Surface plasmon resonance analysis revealed that topoisomerases I and IIalpha bind directly to an immobilized derivative of acetyl-BA. This acetyl-BA derivative interacts with human topoisomerases through high-affinity binding sites yielding K(D) values of 70.6 nM for topoisomerase I and 7.6 nM for topoisomerase IIalpha. Based on our data, we propose that acetyl-BA inhibit topoisomerases I and IIalpha through competition with DNA for binding to the enzyme. Thus, acetyl-BA are a unique class of dual catalytic inhibitors of human topoisomerases I and IIalpha.
- Chen Z et al.
- X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.
- J Mol Biol. 2000; 296: 521-33
- Display abstract
The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.
- Feinberg H, Changela A, Mondragon A
- Protein-nucleotide interactions in E. coli DNA topoisomerase I.
- Nat Struct Biol. 1999; 6: 961-8
- Display abstract
DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA. Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break. The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities. In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides. There are five different binding sites in the complexes, one of which is adjacent to the active site. Two other sites are in the central hole of the protein and may represent general DNA binding regions. The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.
- Mondragon A, DiGate R
- The structure of Escherichia coli DNA topoisomerase III.
- Structure. 1999; 7: 1373-83
- Display abstract
BACKGROUND: DNA topoisomerases are enzymes that change the topology of DNA. Type IA topoisomerases transiently cleave one DNA strand in order to pass another strand or strands through the break. In this manner, they can relax negatively supercoiled DNA and catenate and decatenate DNA molecules. Structural information on Escherichia coli DNA topoisomerase III is important for understanding the mechanism of this type of enzyme and for studying the mechanistic differences among different members of the same subfamily. RESULTS: The structure of the intact and fully active E. coli DNA topoisomerase III has been solved to 3.0 A resolution. The structure shows the characteristic fold of the type IA topoisomerases that is formed by four domains, creating a toroidal protein. There is remarkable structural similarity to the 67 kDa N-terminal fragment of E. coli DNA topoisomerase I, although the relative arrangement of the four domains is significantly different. A major difference is the presence of a 17 amino acid insertion in topoisomerase III that protrudes from the side of the central hole and could be involved in the catenation and decatenation reactions. The active site is formed by highly conserved amino acids, but the structural information and existing biochemical and mutagenesis data are still insufficient to assign specific roles to most of them. The presence of a groove in one side of the protein is suggestive of a single-stranded DNA (ssDNA)-binding region. CONCLUSIONS: The structure of E. coli DNA topoisomerase III resembles the structure of E. coli DNA topoisomerase I except for the presence of a positively charged loop that may be involved in catenation and decatenation. A groove on the side of the protein leads to the active site and is likely to be involved in DNA binding. The structure helps to establish the overall mechanism for the type IA subfamily of topoisomerases with greater confidence and expands the structural basis for understanding these proteins.
- Fiorani P, Amatruda JF, Silvestri A, Butler RH, Bjornsti MA, Benedetti P
- Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity.
- Mol Pharmacol. 1999; 56: 1105-15
- Display abstract
DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.
- Haluska P Jr, Saleem A, Edwards TK, Rubin EH
- Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen.
- Nucleic Acids Res. 1998; 26: 1841-7
- Display abstract
We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I. In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation. Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins. Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay. Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein. Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen. Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.
- Nash HA
- Topological nuts and bolts.
- Science. 1998; 279: 1490-1
- Gmunder H, Kuratli K, Keck W
- In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites.
- Nucleic Acids Res. 1997; 25: 604-11
- Display abstract
A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.
- Shuman S, Bear DG, Sekiguchi J
- Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.
- EMBO J. 1997; 16: 6584-9
- Display abstract
Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.
- Stewart L, Ireton GC, Champoux JJ
- Reconstitution of human topoisomerase I by fragment complementation.
- J Mol Biol. 1997; 269: 355-72
- Display abstract
Human topoisomerase I (topo I, 91 kDa) is composed of four major domains; the unconserved and highly charged "N-terminal" domain (24 kDa), the conserved "core" domain (54 kDa), a poorly conserved and positively charged "linker" region (5 kDa), and the highly conserved "C-terminal" domain (8 kDa) which contains the active site tyrosine at position 723. Here we demonstrate that human topo I activity can be reconstituted by mixing a 58 kDa recombinant core domain (residues Lys175 to Ala659) with any one of a series of recombinant C-terminal fragments that range in size from 12 kDa (linker and C-terminal domains, residues Leu658 to Phe765) to 6.3 kDa (C-terminal domain residues Gln713 to Phe765). The C-terminal fragments bind tightly to the core domain, forming a 1:1 complex that is stable irrespective of ionic strength (0.01 to 1 M). The reconstituted enzymes are active only over a relatively narrow range of salt concentrations (25 to 200 mM KCl) as compared to the intact topo70 enzyme (missing the N-terminal domain). Under physiological conditions (150 mM KCl and 10 mM Mg2+) they are much more distributive in their mode of action than topo70. The reconstituted enzyme binds DNA with an affinity that is approximately 20-fold lower than that of the intact topo70. In addition, the cleavage/religation equilibrium of the reconstituted enzyme appears to be biased towards religation relative to that of the intact enzyme. Despite differences in the cleavage/religation equilibrium and affinity for DNA, the reconstituted and intact enzymes have identical sequence specificities for the cleavage of duplex DNA or suicide cleavage of oligonucleotide substrates.
- Kampranis SC, Maxwell A
- Conversion of DNA gyrase into a conventional type II topoisomerase.
- Proc Natl Acad Sci U S A. 1996; 93: 14416-21
- Display abstract
DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperature-sensitive phenotype of a parCts mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits and provide an insight into the mechanism of type II topoisomerases.
- Sandri MI et al.
- p53 regulates the minimal promoter of the human topoisomerase IIalpha gene.
- Nucleic Acids Res. 1996; 24: 4464-70
- Display abstract
DNA topoisomerase IIalpha is an essential enzyme for chromosome segregation during mitosis. Consistent with a cell division-specific role, the expression of the topoisomerase IIalpha gene is strongly influenced by the proliferation status of cells. The p53 protein is one of the most important regulators of cell cycle progression in mammals, with an apparent dual role in the induction of cell cycle arrest following cytotoxic insults and in the regulation of the apoptotic cell death pathway. We have analysed whether p53 plays a role in regulating expression of the human topoisomerase IIalpha gene. We show that wild-type, but not mutant, p53 is able to decrease substantially the activity of the full length topoisomerase IIalpha gene promoter. Using a series of constructs comprising various deleted or mutated versions of the promoter lacking critical cis-acting elements, we show that this p53-specific regulation of the topoisomerase IIalpha promoter is independent of all characterised transcription factor binding sites and is directed at the minimal gene promoter. We conclude that expression of wild-type p53 induces downregulation of the human topoisomerase IIalpha promoter by acting on the basal transcription machinery. These findings implicate topoisomerase II as one of the downstream targets for p53-dependent regulation of cell cycle progression in human cells.
- Wang JC
- DNA topoisomerases.
- Annu Rev Biochem. 1996; 65: 635-92
- Display abstract
The various problems of disentangling DNA strands or duplexes in a cell are all rooted in the double-helical structure of DNA. Three distinct subfamilies of enzymes, known as the DNA topoisomerases, have evolved to solve these problems. This review focuses on work in the past decade on the mechanisms and cellular functions of these enzymes. Newly discovered members and recent biochemical and structural results are reviewed, and mechanistic implications of these results are summarized. The primary cellular functions of these enzymes, including their roles in replication, transcription, chromosome condensation, and the maintenance of genome stability, are then discussed. The review ends with a summary of the regulation of the cellular levels of these enzymes and a discussion of their association with other cellular proteins.
- Gomez-Eichelmann MC, Camacho-Carranza R
- [DNA supercoiling and topoisomerases in Escherichia coli].
- Rev Latinoam Microbiol. 1995; 37: 291-304
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The chromosomal DNA of all cells is under helical tension or supercoiling. There are two classes of DNA supercoiling: plectonemic and toroidal. Plectonemic supercoiling is generated by the action of DNA topoisomerases, while toroidal supercoiling is generated by DNA-protein interactions and by topoisomerase activitities. DNA supercoiling plays an important role in replication, repair, recombination, transposition and transcription. DNA topoisomerases type I are ATP-independent enzymes that cut one DNA strand and relax supercoiled molecules. DNA topoisomerases type II requiere ATP, cut both DNA strands and supercoil relaxed molecules. All organisms have more than one topoisomerase of each, type I and type II. Escherichia coli has two topoisomerases type I: topoisomerase I and topoisomerase III and two topoisomerases type II: topoisomerase II or gyrase and topoisomerase IV. In this review we discuss the concept of DNA supercoiling and present current knowledge on E. coli DNA topoisomerases.
- Lima CD, Wang JC, Mondragon A
- Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I.
- Nature. 1994; 367: 138-46
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The three-dimensional structure of the 67K amino-terminal fragment of Escherichia coli DNA topoisomerase I has been determined to 2.2 A resolution. The polypeptide folds in an unusual way to give four distinct domains enclosing a hole large enough to accommodate a double-stranded DNA. The active-site tyrosyl residue, which is involved in the transient breakage of a DNA strand and the formation of a covalent enzyme-DNA intermediate, is present at the interface of two domains. The structure suggests a plausible mechanism by which E. coli DNA topoisomerase I and other members of the same DNA topoisomerase subfamily could catalyse the passage of one DNA strand through a transient break in another strand.
- Sharma A, Hanai R, Mondragon A
- Crystal structure of the amino-terminal fragment of vaccinia virus DNA topoisomerase I at 1.6 A resolution.
- Structure. 1994; 2: 767-77
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BACKGROUND: Vaccinia virus, a cytoplasmically-replicating poxvirus, encodes a type I DNA topoisomerase that is biochemically similar to eukaryotic-like DNA topoisomerases I, and which has been widely studied as a model topoisomerase. It is the smallest topoisomerase known and is unusual in that it is resistant to the potent chemotherapeutic agent camptothecin. RESULTS: The crystal structure of a 9 kDa amino-terminal fragment of vaccinia virus DNA topoisomerase I has been determined at 1.6 A resolution. The fragment forms a five-stranded, antiparallel beta-sheet with two short alpha-helices and connecting loops. Residues that are conserved between all eukaryotic-like type I topoisomerases are not clustered in particular regions of the structure. CONCLUSIONS: This is the first atomic structure of any region of a eukaryotic-like DNA topoisomerase I. It has provided insights into the structural bases of the phenotypes of some single-site mutants of the intact topoisomerase. The structure has enabled us to study the interactions within a well-folded protein fragment and the camptothecin resistance of the viral topoisomerase.
- Samuel M, Zhu CX, Villanueva GB, Tse-Dinh YC
- Effect of zinc removal on the conformation of Escherichia coli DNA topoisomerase I.
- Arch Biochem Biophys. 1993; 300: 302-8
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Escherichia coli DNA topoisomerase I contains three Zn(II) in each enzyme molecule required for relaxation of negatively supercoiled DNA. Apoenzymes were prepared from both the intact topoisomerase (M(r) 97,000) and the truncated active form top85 (M(r) 85,000) that lacks the carboxyl terminal domain but still contains the three Zn(II). Fluorescence and circular dichroism spectroscopy were used to compare the apoenzymes with topoisomerase and top85 reconstituted with Zn2+. The results indicated structural changes affecting the environment of the tryptophan residues and increasing the alpha-helical and beta-sheets content of the protein occurred upon zinc removal. These structural changes probably account for the loss of enzyme activity.
- Trask DK, DiDonato JA, Muller MT
- Rapid detection and isolation of covalent DNA/protein complexes: application to topoisomerase I and II.
- EMBO J. 1984; 3: 671-6
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A rapid and simple method has been developed which allows detection and isolation of covalent DNA/protein adducts. The method is based upon the use of an ionic detergent, SDS, to neutralize cationic sites of weakly bound proteins thereby resulting in their dissociation off the helix. Proteins tightly or covalently bound to DNA that are not dissociable by SDS, result in the precipitation of the DNA fragment by the addition of KCl; however, free nucleic acid does not precipitate. The method is particularly useful as an analytical tool to titrate the binding of prototypic covalent binding proteins, topoisomerase I and II; thus, quantitation of topoisomerase activity is possible under defined conditions. As an analytical tool the method can be used as a general assay in the purification of as yet unidentified topoisomerases or other activities that bind DNA covalently. Moreover, the technology can be adapted for use in a preparative mode to separate covalent complexes from free DNA in a single step.