First functional characterization of a singly expressed bacterial type II topoisomerase: The enzyme from Mycobacterium tuberculosis

doi:10.1016/j.bbrc.2006.07.017

Biochemical and Biophysical Research Communications

Volume 348, Issue 1, 15 September 2006, Pages 158-165

https://doi.org/10.1016/j.bbrc.2006.07.017 Get rights and content

Abstract

Genome deciphering revealed that Mycobacterium tuberculosis encodes a single type II topoisomerase contrary to common bacteria harboring two type II topoisomerases (DNA gyrase and topoisomerase IV). Functions of the M. tuberculosis type II topoisomerase were explored after cloning and expressing the subunits encoding genes in Escherichia coli. M. tuberculosis type II topoisomerase supercoiled relaxed pBR322 with a specific activity close to that of DNA gyrases of common bacteria whereas it exhibited DNA relaxation and formation of cleavable complexes with activities significantly higher than other DNA gyrases. Intermolecular passage activity evaluated by the decatenation of kinetoplast DNA was 25-fold lower than that of the topoisomerase IV from Streptococcus pneumoniae, but was markedly higher than that of the E. coli gyrase. Overall, the type II topoisomerase of M. tuberculosis exhibits classical polyvalent activities of DNA gyrase for supercoiling but enhanced relaxation, cleavage, and decatenation activities.

Section snippets

Materials and methods

Substrates, inhibitors, and reagents. Supercoiled plasmid pBR322 DNA was purchased from Roche (Roche Diagnostics, Meylan Cedex, France), relaxed plasmid pBR322 DNA from John Innes Enterprises, Ltd. (Norwich Research Park, Colney, Norwich, UK), and kinetoplast DNA (kDNA) from Topogen (Denver, CO, USA). The following chemicals were used: magnesium acetate and manganese chloride (Sigma–Aldrich Chimie, Saint Quentin Fallavier, France), calcium chloride (Prolabo, Paris, France), and potassium

Intramolecular DNA processing activities of the type II topoisomerase of M. tuberculosis

The recombinant type II M. tuberculosis topoisomerase supercoiled relaxed pBR322 DNA gyrase with a specific activity reaching 2 × 10⁴ U/mg (Fig. 1). Supercoiling was observed for ATP concentrations ranging from 0.5 to 8 mM, but was reduced at higher concentrations. Partial supercoiling was observed when ATP was replaced by dATP or dTTP whereas no supercoiling was observed for either CTP, GTP or other dNTPs. Mg²⁺ was the cation required for DNA supercoiling with an optimum concentration of 6 mM (Fig.

Discussion

Mycobacterium tuberculosis and a few other bacteria harbor only one type II topoisomerase in contrast with E. coli and other common bacteria which harbor two type II topoisomerases, DNA gyrase and topoisomerase IV. This situation raises interesting questions as to the nature of the activities of the single type II topoisomerases, and the present study is the first characterization of such an enzyme. Since, we recently succeeded in producing highly purified recombinant type II topoisomerase of

Acknowledgments

We thank Wladimir Sougakoff and Claudine Mayer for helpful discussion, and Xiao-Su Pan for the S. pneumoniae topoisomerases. This work was supported by grants from INSERM (EMI 0004), the University of Paris (UPRES 1541), the Foundation pour la Recherche Médicale, and the Association Française Raoul Follereau. Work in the Fisher lab was supported by the Biotechnology and Biological Sciences Research Council, UK.

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DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection. Gyrase hydrolyzes ATP to add negative supercoils to DNA using a strand passage mechanism that has been investigated using biophysical and biochemical approaches. To analyze the dynamics of substeps leading to strand passage, single-molecule rotor bead tracking (RBT) has been used previously to follow real-time supercoiling and conformational transitions in Escherichia coli (EC) gyrase. However, RBT has not yet been applied to gyrase from other pathogenically relevant bacteria, and it is not known whether substeps are conserved across evolutionarily distant species. Here, we compare gyrase supercoiling dynamics between two evolutionarily distant bacterial species, MTB and EC. We used RBT to measure supercoiling rates, processivities, and the geometries and transition kinetics of conformational states of purified gyrase proteins in complex with DNA. Our results show that E. coli and MTB gyrases are both processive, with the MTB enzyme displaying velocities ∼5.5× slower than the EC enzyme. Compared with EC gyrase, MTB gyrase also more readily populates an intermediate state with DNA chirally wrapped around the enzyme, in both the presence and absence of ATP. Our substep measurements reveal common features in conformational states of EC and MTB gyrases interacting with DNA but also suggest differences in populations and transition rates that may reflect distinct cellular needs between these two species.
Thermus thermophilus Argonaute Functions in the Completion of DNA Replication
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In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
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Tyrosine at the 577 position and two arginines at 691 and 745 positions in CTD of GyrA are the residues responsible for DNA binding [36]. However, TopIV from S. pneumoniae is a more powerful decatenase than Mtb gyrase [26]. Switching on to supercoiling activity of Mtb gyrase, it exhibits less potential than the E. coli counterpart due to the deficiency of a ‘control element’ in the CTD of GyrA [37], unlike E. coli.
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First functional characterization of a singly expressed bacterial type II topoisomerase: The enzyme from Mycobacterium tuberculosis

Abstract

Section snippets

Materials and methods

Intramolecular DNA processing activities of the type II topoisomerase of M. tuberculosis

Discussion

Acknowledgments

Biochem. Biophys. Acta

Cell

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Structure (Camb)

DNA topoisomerases: structure, function, and mechanism

Annu. Rev. Biochem.

Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases

Annu. Rev. Biophys. Biomol. Struct.

The C-terminal domain of DNA gyrase A adopts a DNA-bending beta-pinwheel fold

Proc. Natl. Acad. Sci. USA