Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase–helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein–protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.     

The interaction between coumarin drugs and DNA gyrase

The coumarin group of antibiotics have as their target the bacterial enzyme DNA gyrase. The drugs bind to the B subunit of gyrase and inhibit DNA supercoiling by blocking the ATPase activity. Recent data show that the binding site for the drugs lies within the N-terminal part of the B protein, and individual amino acids involved in coumarin interaction are being identified. The mode of inhibition of the gyrase ATPase reaction by coumarins is unlikely to be simple competitive inhibition, and the drugs may act by stabilizing a conformation of the enzyme with low affinity for ATP.     

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.     

GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase

DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase–DNA covalent complexes leading to double-strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (GyrI) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. GyrI reduces intrinsic as well as toxin-stabilized gyrase–DNA covalent complexes. Furthermore, GyrI reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, GyrI is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems.     

Computer-aided identification of novel 3,5-substituted rhodanine derivatives with activity against Staphylococcus aureus DNA gyrase

Methicillin resistant Staphylococcus aureus (MRSA) is among the major drug resistant bacteria that persist in both the community and clinical settings due to resistance to commonly used antimicrobials. This continues to fuel the need for novel compounds that are active against this organism. For this purpose we have targeted the type IIA bacterial topoisomerase, DNA gyrase, an essential enzyme involved in bacterial replication, through the ATP-dependent supercoiling of DNA. The virtual screening tool Shape Signatures was applied to screen a large database for agents with shape similar to Novobiocin, a known gyrase B inhibitor. The binding energetics of the top hits from this initial screen were further validated by molecular docking. Compounds with the highest score on available crystal structure of homologous DNA gyrase from Thermus thermophilus were selected. From this initial set of compounds, several rhodanine-substituted derivatives had the highest antimicrobial activity against S. aureus, as determined by minimal inhibitory concentration assays, with Novobiocin as the positive control. Further activity validation of the rhodanine compounds through biochemical assays confirmed their inhibition of both the supercoiling and the ATPase activity of DNA gyrase. Subsequent docking and molecular dynamics on the crystal structure of DNA gyrase from S. aureus when it became available, provides further rationalization of the observed biochemical activity and understanding of the receptor–ligand interactions. A regression model for MIC prediction against S. aureus is generated based on the current molecules studied as well as other rhodanines derivatives found in the literature.     

A 4.2 kDa synthetic peptide as a potential probe to evaluate the antibacterial activity of coumarin drugs.

The coumarin antibiotics are potent inhibitors of DNA replication whose target is the enzyme DNA gyrase, an ATP-dependent bacterial type II topoisomerase. The coumarin drugs inhibit gyrase action by competitive binding to the ATP-binding site of DNA gyrase B protein. The production of new biologically active products has stimulated additional studies on coumarin-gyrase interactions. In this regard, a 4.2 kDa peptide mimic of DNA gyrase B protein from Escherichia coli has been designed and synthesized. The peptide sequence includes the natural fragment 131-146 (coumarin resistance-determining region) and a segment containing the gyrase-DNA interaction region (positions 753-770). The peptide mimic binds to novobiocin (Ka = 1.4+/-0.3 x 10(5) M(-1)), plasmid (Ka = 1.6+/-0.5 x 10(6) M(-1)) and ATP (Ka = 1.9+/-50.4 x 10(3) M(-1)), results previously found with the intact B protein. On the other hand, the binding to novobiocin was reduced when a mutation of Arg-136 to Leu-136 was introduced, a change previously found in the DNA gyrase B protein from several coumarin-resistant clinical isolates of Escherichia coli In contrast, the binding to plasmid and to ATP was not altered. These results suggest that synthetic peptides designed in a similar way to that described here could be used as mimics of DNA gyrase in studies which seek a better understanding of the ATP, as well as coumarin, binding to the gyrase and also the mechanism of action of this class of antibacterial drugs.     

Dissection of the nucleotide cycle of B. subtilis DNA gyrase and its modulation by DNA

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. While all type II DNA topoisomerases relax supercoiled DNA, DNA gyrase is the only enyzme that introduces negative supercoils into DNA at the expense of ATP hydrolysis. We present here a biophysical characterization of the nucleotide cycle of DNA gyrase from Bacillus subtilis, both in the absence and presence of DNA. B. subtilis DNA gyrase is highly homologous to its well-studied Escherichia coli counterpart, but exhibits unique mechanistic features. The active heterotetramer of B. subtilis DNA gyrase is formed by mixing the GyrA and GyrB subunits. GyrB undergoes nucleotide-induced dimerization and is an ATP-operated clamp. The intrinsic ATPase activity of gyrase is stimulated tenfold in the presence of plasmid DNA. However, in contrast to the E. coli homolog, the rate-limiting step in the nucleotide cycle of B. subtilis GyrB is ATP hydrolysis, not product dissociation or an associated conformational change. Furthermore, there is no cooperativity between the two DNA and ATP binding sites in B. subtilis DNA gyrase. Nevertheless, the enzyme is as efficient in negative supercoiling as the E. coli DNA gyrase. Our results provide evidence that the evolutionary goal of efficient DNA supercoiling can be realized by similar architecture, but differences in the underlying mechanism. The basic mechanistic features are conserved among DNA gyrases, but the kinetics of individual steps can vary significantly even between closely related enzymes. This suggests that each topoisomerase represents a different solution to the complex reaction sequence in DNA supercoiling.     

Reverse gyrase has heat-protective DNA chaperone activity independent of supercoiling

Hyperthermophilic organisms must protect their constituent macromolecules from heat-induced degradation. A general mechanism for thermoprotection of DNA in active cells is unknown. We show that reverse gyrase, the only protein that is both specific and common to all hyperthermophiles, reduces the rate of double-stranded DNA breakage ~8-fold at 90°C. This activity does not require ATP hydrolysis and is independent of the positive supercoiling activity of the enzyme. Reverse gyrase has a minor nonspecific effect on the rate of depurination, and a major specific effect on the rate of double-strand breakage. Using electron microscopy, we show that reverse gyrase recognizes nicked DNA and recruits a protein coat to the site of damage through cooperative binding. Analogously to molecular chaperones that assist unfolded proteins, we found that reverse gyrase prevents inappropriate aggregation of denatured DNA regions and promotes correct annealing. We propose a model for a targeted protection mechanism in vivo in which reverse gyrase detects damaged DNA and acts as a molecular splint to prevent DNA breakage in the vicinity of the lesion, thus maintaining damaged DNA in a conformation that is amenable to repair.     

A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima

We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B). The deduced protein is composed of 636 aa with a calculated molecular mass of 72 415 Da. It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE). Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV. This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability. No gyrA-like gene has been found near top2B. A gene coding for a transaminase B-like protein was found in the upstream region of top2B.     

Reverse gyrase of Sulfolobus: purification to homogeneity and characterization

By using hydrophobic interaction as the first chromatographic stage, we purified to homogeneity reverse gyrase, an ATP-dependent DNA topoisomerase I, isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldrius. This procedure allowed quick and complete separation of reverse gyrase from nucleases and DNA binding proteins present in Sulfolobus. The final product was revealed, by SDS-PAGE, as a unique band with an apparent molecular mass of 128 kDa, and the amino acid composition was determined. Western blotting experiments with antibodies raised against reverse gyrase indicate that no proteolysis occurred during the purification course. Gel filtration and sedimentation data gave a Stokes radius of 42 A and a sedimentation coefficient of 5.7 S, suggesting a monomeric structure for the native enzyme which was confirmed by electron microscopy. Finally, pure reverse gyrase in a monomeric state was still able to promote positive supercoiling of the DNA.     

Functional characterisation of mycobacterial DNA gyrase: an efficient decatenase

A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli gyrase. In contrast to E.coli gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg²⁺ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K_0.5 and k_cat of 0.68 mM and 0.39 s^–1, respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.     

Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.     

The DNA dependence of the ATPase activity of DNA gyrase

We have studied the ATPase activity of DNA gyrase both in the absence and presence of DNA. In the absence of DNA we show that the gyrase B protein alone has a very low level of ATPase activity which can be increased many-fold by pretreatment of the B protein with heat or urea. When both the gyrase A protein and linear DNA are also present, the ATPase activity of the untreated B protein is greatly stimulated. We find that the extent of stimulation is dependent upon the length of the DNA but largely independent of DNA sequence. DNA molecules greater than 100 base pairs in length are much more effective in stimulating the gyrase ATPase than those of 70 base pairs or less, although short DNA molecules will stimulate the ATPase at high concentrations. The behavior of long and short DNA molecules with respect to ATPase stimulation is also reflected in their abilities to bind DNA gyrase. To account for these data we propose a model for the interaction of gyrase with ATP and DNA in which ATP hydrolysis requires the binding of DNA to two sites on the enzyme.     

Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.     

Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage

DNA gyrase catalyses DNA supercoiling by passing one segment of DNA (the T segment) through another (the G segment) in a reaction coupled to the binding and hydrolysis of ATP. The N-terminal domains of the gyrase B dimer constitute an ATP-operated clamp that is proposed to capture the T segment during the DNA supercoiling reaction. We have locked this clamp in the closed conformation using the non-hydrolysable ATP analogue ADPNP (5'-adenylyl beta,gamma-imidodiphosphate). The clamp-locked enzyme is able to bind and cleave DNA, albeit at a reduced level. Although the locked enzyme is not capable of carrying out DNA supercoiling, it can catalyse limited DNA relaxation, consistent with the ability to complete one strand passage event per enzyme molecule via entry of the T segment through the exit gate of the enzyme. The DNA-protein complex of the clamp-locked enzyme has a conformation that differs from the normal positively wrapped conformation of the gyrase-DNA complex. These experiments confirm the role of the ATP-operated clamp in the strand-passage reactions of gyrase and suggest a model for the interaction of DNA with gyrase in which a conformation with the T segment in equilibrium across the DNA gate can be achieved via T-segment entry through the ATP-operated clamp or through the exit gate.     

Mechanism of quinolone inhibition of DNA gyrase. Appearance of unique norfloxacin binding sites in enzyme-DNA complexes

As a means of gaining additional information on the topoisomerase-mediated cytotoxicity induced by a variety of antibacterial and antitumor compounds we have examined the interaction of the quinolone anti-bacterial agent, norfloxacin, with the bacterial topoisomerase, DNA gyrase. Membrane filtration and spin-column techniques were used to study the binding of [3H]norfloxacin to purified plasmid DNA, DNA gyrase, and complexes formed by adding gyrase to different forms of plasmid DNA. Consistent with previous results (Shen, L. L., and Pernet, A. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 301-311) little [3H]norfloxacin binds to reconstituted gyrase, but significant levels of drug bind nonspecifically to relaxed DNA. However, when DNA and gyrase are incubated together additional norfloxacin binding sites are detectable. These complex-dependent sites are distinguishable from those sites involved in nonspecific DNA binding in that the complex-dependent sites are saturable and they retain bound norfloxacin after centrifuging the complex through a spin column. In addition, extent of binding is influenced by the topological state of DNA used to form the complex. The complex-dependent norfloxacin binding sites are likely involved in the inhibition of the enzyme since saturation of these sites occurs in the same norfloxacin concentration range as the inhibition of DNA supercoiling activity. Moreover, there is a close correlation of norfloxacin-induced DNA breakage with levels of norfloxacin bound to complexes of gyrase and relaxed DNA. These findings provide the first direct correlation of quinolone binding with inhibition of enzyme activity and induction of DNA breakage, and they suggest that the inhibition of DNA gyrase by norfloxacin occurs as a result of binding to a site which appears after the formation of a gyrase-DNA complex.     

Unique features of apicoplast DNA gyrases from Toxoplasma gondii and Plasmodium falciparum

Background

DNA gyrase, an enzyme once thought to be unique to bacteria, is also found in some eukaryotic plastids including the apicoplast of Apicomplexa such as Plasmodium falciparum and Toxoplasma gondii which are important disease-causing organisms. DNA gyrase is an excellent target for antibacterial drugs, yet such antibacterials seem ineffective against Apicomplexa. Characterisation of the apicoplast gyrases would be a useful step towards understanding why this should be so. While purification of active apicoplast gyrase has proved impossible to date, in silico analyses have allowed us to discover differences in the apicoplast proteins. The resulting predicted structural and functional differences will be a first step towards development of apicoplast-gyrase specific inhibitors.

Results

We have carried out sequence analysis and structural predictions of the enzymes from the two species and find that P. falciparum gyrase lacks a GyrA box, but T. gondii may retain one. All proteins contained signal/transport peptides for localization to the apicoplast but T. gondii Gyrase B protein lacks the expected hydrophobic region. The most significant difference is in the GyrA C-terminal domain: While the cores of the proteins, including DNA binding and cleavage regions are essentially unchanged, both apicoplast gyrase A proteins have C-terminal domains that are significantly larger than bacterial counterparts and are predicted to have different structures.

Conclusion

The apicoplast gyrases differ significantly from bacterial gyrases while retaining similar core domains. T. gondii Gyrase B may have an unusual or inefficient mechanism of localisation to the apicoplast. P.falciparum gyrase, lacks a GyrA box and is therefore likely to be inefficient in DNA supercoiling. The C-terminal domains of both apicoplast Gyrase A proteins diverge significantly from the bacterial proteins. We predict that an additional structural element is present in the C-terminal domain of both apicoplast Gyrase A proteins, including the possibility of a β-pinwheel with a non-canonical number of blades. These differences undoubtedly will affect the DNA supercoiling mechanism and have perhaps evolved to compensate for the lack of Topoisomerase IV in the apicoplast. These data will be useful first step towards further characterisation and development of inhibitors for apicoplast gyrases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0416-9) contains supplementary material, which is available to authorized users.