Phylogenomics of type II DNA topoisomerases

Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.     

Computational analysis of the chiral action of type II DNA topoisomerases

It was found recently that bacterial type II DNA topoisomerase, topo IV, is much more efficient in relaxing (+) DNA supercoiling than (-) supercoiling. This means that the DNA-enzyme complex is chiral. This chirality can appear upon binding the first segment that participates in the strand passing reaction (G segment) or only after the second segment (T segment) joins the complex. The former possibility is analyzed here. We assume that upon binding the enzyme, the G segment forms a part of left-handed helical turn. This model is an extension of the hairpin model introduced earlier to explain simplification of DNA topology by these enzymes. Using statistical-mechanical simulation of DNA properties, we estimated different consequences of the model: (1) relative rates of relaxation of (+) and (-) supercoiling by the enzyme; (2) the distribution of positions of the G segment in supercoiled molecules; (3) steady-state distribution of knots in circular molecules created by the topoisomerase; (4) the variance of topoisomer distribution created by the enzyme; (5) the effect of (+) and (-) supercoiling on the binding topo II with G segment. The simulation results are capable of explaining nearly all available experimental data, at least semiquantitatively. A few predictions obtained in the model analysis can be tested experimentally.     

Anaphase chromatid motion: involvement of type II DNA topoisomerases.

Sister chromatids are topologically intertwined at the onset of anaphase: their segregation during anaphase is known to require strand-passing activity by type II DNA topoisomerase. We propose that the removal of the intertwinings involves at the same time the traction of the mitotic spindle and the activity of topoisomerases. This implies that the velocity of the chromatids is compatible with the kinetic constraints imposed by the enzymatic reaction. We show that the greatest observed velocities (about 0.1 microns s-1) are close to the theoretical upper bound compatible with both the diffusion rate (calculated here within a probabilistic model) and the measured reaction rate of the enzyme.     

Epitope distribution of randomly established monoclonal antibodies against human type II DNA topoisomerases

The epitopes of about 100 monoclonal antibodies against human type II DNA topoisomerase were mapped along the enzyme molecules. Although they were randomly and independently established, epitope sites were unevenly distributed the toward N-terminal or C-terminal region. We suggest that the central catalytic domain is hidden inside the molecule and inaccessible to the antigen recognition sites. Using antibodies, we demonstrate the distinct localization of isoforms of Topo II in cultured cells. Some particularly useful antibodies are listed.     

Overexpression of type I topoisomerases sensitizes yeast cells to DNA damage

DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.     

A quantitative decatenation assay for type II topoisomerases

Type II topoisomerases catalyze decatenation of the catenated network of kinetoplast DNA [J. C. Marini, K. G. Miller, and P. T. Englund (1980) J. Biol. Chem. 255, 4976-4979]. The individual DNA circles and small catenanes produced during the decatenation reaction can be separated from the large network of substrate DNA by 5 min centrifugation at 13,000g and quantitated. The appearance of these decatenated DNA molecules which appear in the supernatant first showed a lag, whose duration depended on the enzyme concentration, and then increased linearly with time until it reached a plateau. The slope of the linear part of the kinetic curve was directly proportional to the enzyme concentration, whether a purified or crude preparation of type II topoisomerase from mammalian cells was used. These findings led us to a rapid quantitative assay of type II topoisomerases not involving electrophoresis. The method was developed with purified enzyme but was also useful for assay of the activity in crude extracts. Surprisingly, the type I topoisomerase, even when present in large excess, failed to decatenate the nicked DNA circles often present in the kinetoplast DNA. This renders the assay virtually free from interference by type I enzyme. The method is sensitive and allowed quantitative estimation of the enzyme activity present in the crude extracts corresponding to that derived from 500 to 700 cultured mammalian cells. Since various type II topoisomerases from procaryotic, eucaryotic, and viral sources decatenate kinetoplast DNA and generate similar DNA products, the assay method is likely to be generally applicable.     

Bacterial type II topoisomerases as targets for antibacterial drugs

Bacterial type II DNA topoisomerases are essential enzymes for correct genome functioning and cell growth. Gyrase is responsible for maintaining negative supercoiling of bacterial chromosome, whereas topoisomerase IV acts in disentangling daughter chromosomes following replication. Type II DNA topoisomerases possess an ATP binding site, which can be treated as a target for antibacterial drugs. Resolving crystal structures of protein fragments consisting of an ATP binding site complexed with ADPNP/antibiotics have proven to be valuable for the understanding of the mode of action of existing antibacterial agents and presented new possibilities for novel drug design. Coumarins, quinolones and cyclothialidines are diverse group of antibiotics that interfere with type II DNA topoisomerases, however their mode of action is different. Recently a new class of antibiotics, simociclinones, was characterized. Their mechanism of action towards gyrase is entirely distinct from already known modes of action, therefore demonstrating the potential for development of novel anti-bacterial agents.     

Novel pyrazole derivatives as potent inhibitors of type II topoisomerases. Part 1: synthesis and preliminary SAR analysis

In an attempt to search for a new class of antibacterial agents, we have discovered a series of pyrazole analogs that possess good antibacterial activity for Gram-positive and Gram-negative organisms via inhibition of type II bacterial topoisomerases. We have investigated the structure-activity relationships of this series, with an emphasis on the length and conformation of the linker. This work led to the identification of tetrahydroindazole analogs, such as compound 1, as the most potent class of compounds.     

Functional interactions of DNA topoisomerases with a human replication origin

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.     

Molecular interactions between Wells-Dawson type polyoxometalates and human serum albumin

Binding human serum albumin (HSA) of three polyoxometalates (POMs) with the Wells-Dawson structure, alpha(2)-[P2W17O61]10- (abbreviated as alpha(2)-P2W17) and two of its metal-substituted derivatives, alpha(2)-[NiP2W17O61]8- and alpha(2)-[CuP2W17O61]8- (alpha(2)-P2W17Ni and alpha(2)-P2W17Cu, respectively) was studied in an aqueous medium at pH 7.5. Fluorescence quenching, circular dichroism (CD), thermal denaturation, and isothermal titration calorimetry (ITC) were used for this purpose. The results were compared with those obtained previously with the Keggin structure POM, [H2W12O40]6- (H2W12), and the wheel-shaped structure, [NaP5W30O110]14- (P5W30). All these POMs bind HSA mainly by electrostatic interactions. Comparison of the physical characteristics and HSA interaction parameters for the POMs of the present work and those studied previously showed that the overall charge of the clusters is not the single parameter governing the binding process and its consequences. In contrast, besides the influences of the structure, the dimension and/or weight of the POMs, the results have permitted highlighting of the importance of each POM atomic composition for its binding behavior.     

Molecular cloning and binding properties of the human type II activin receptor.

A full-length cDNA for the type II human activin receptor was cloned by hybridization from a human testis cDNA library. The sequence encodes a 513 amino acid protein that is 99% identical, at the amino acid level, with the mouse type II activin receptor. The type II human activin receptor consists of an extracellular domain that specifically binds activin A with a Kd of 360 pM, a single-membrane spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity.     

Ungeremine effectively targets mammalian as well as bacterial type I and type II topoisomerases

From the methanol extract of the bulbs of Pancratium illyricum L., three phenanthridine type alkaloids, ungeremine (1), (-)-lycorine (2) and (+)-vittatine (3) were isolated. For the evaluation of their anticancer and antibacterial potential, compounds 1-3 were tested against human (I, IIα) and bacterial (IA, IV) topoisomerases. Our data demonstrated that ungeremine impairs the activity of both, human and bacterial topoisomerases. Remarkably, ungeremine was found to largely increments the DNA cleavage promoted by bacterial topoisomerase IA, a new target in antimicrobial chemotherapy.     

Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases

Antibacterial fluoroquinolones trap a cleavage complex of gyrase and topoisomerase (topo) IV inducing site-specific DNA breakage within a bent DNA gate engaged in DNA transport. Despite its importance for drug action and in revealing potential sites of topoisomerase catalysis, the mechanism of DNA selectivity is poorly understood. To explore its functional basis, we generated mutant versions of the strongly cleaved E-site and used a novel competitive assay to examine their gemifloxacin-mediated DNA breakage by Streptococcus pneumoniae topo IV and gyrase. Parallel studies of Ca²⁺-induced cleavage distinguished ‘intrinsic recognition’ of DNA cleavage sites by topo IV from drug-induced preferences. Analysis revealed strong enzyme-determined requirements for −4G, −2A and −1T bases preceding the breakage site (between −1 and +1) and enzyme-unique or degenerate determinants at −3, plus drug-specific preferences at +2/+3 and for +1 purines associated with drug intercalation. Similar cleavage rules were seen additionally at the novel V-site identified here in ColE1-derived plasmids. In concert with DNA binding data, our results provide functional evidence for DNA, enzyme and drug contributions to DNA cleavage at the gate, suggest a mechanism for DNA discrimination involving enzyme-induced DNA bending/helix distortion and cleavage complex stabilization and advance understanding of fluoroquinolones as important cleavage-enhancing therapeutics.     

The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.     

Binding of two DNA molecules by type II topoisomerases for decatenation

Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.     

Molecular assembly of recombinant chicken type II collagen in the yeast Pichia pastoris

Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen(n CCII), recombinant peptide containing n CCII tolerogenic epitopes(CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2 A1 c DNA. As recombinant CCII(r CCII) might avoid potential pathogenic virus contamination during n CCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on r CCII triple helix molecular assembly. We constructed p C-and p N-procollagen(without N-or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115(his4, Mut+) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both p C-and p N-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight p C-or p N-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen p Cα1(II) can induce collagen-induced arthritis(CIA) rat model, which seems to be as effective as the current standard n CCII. Notably, protease digestion assays showed that r CCII could assemble in the absence of C-and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for r CCII expression and folding.