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.     

DNA-DNA gyrase complex: the wrapping of the DNA duplex outside the enzyme.

Digestion of the complex between double-stranded DNA and M. luteus or E. coli DNA gyrase with staphylococcal nuclease gives a 143 ± 3 base pair DNA fragment containing no single-chain scissions. Digestion of the same complex with bovine pancreatic DNAase I gives six discernible single-stranded DNA bands upon electrophoresis of the product in a denaturing gel. The lengths of these fragments, in number of nucleotides, are measured to be 47 ± 1, 57 ± 1, 67 ± 1, 77 ± 1, 86 ± 1 and 96 ± 1, respectively. These results support the notion that in the DNA-gyrase complex, a segment(s) of the DNA helix is wrapped around the enzyme. The wrapping of the DNA around the enzyme has been proposed previously based on the observation that in the absence of ATP, the linking number of a duplex DNA ring covalently closed by ligase in the presence of bound gyrase is higher than in the absence of gyrase (Liu and Wang, 1978). The coiling of DNA around the enzyme in the complex is believed to be intimately related to the ATP-dependent negative supercoiling of covalently closed duplex DNA ring by DNA gyrase. It has also been observed that digestion of pure double-stranded DNA by pancreatic DNAase I in the presence of calcium phosphate precipitate or solid hydroxylapatite gives a ladder of single-stranded DNA fragments of integral multiples of 10 nucleotides. This finding suggests that such a pancreatic DNAase I cleavage pattern is indicative of a DNA duplex lying on the outside of a surface.     

In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites.

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.     

Evidence for a conformational change in the DNA gyrase-DNA complex from hydroxyl radical footprinting.

We have used the technique of hydroxyl radical footprinting to probe the complex between DNA gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. We find that gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl-beta,gamma-imidodiphosphate or a quinolone drug causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. The significance of these results for the mechanism of DNA supercoiling by gyrase is discussed.     

Nucleotide binding to DNA gyrase causes loss of DNA wrap

DNA gyrase negatively supercoils DNA in a reaction coupled to the binding and hydrolysis of ATP. Limited supercoiling can be achieved in the presence of the non-hydrolysable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (ADPNP). In order to negatively supercoil DNA, gyrase must wrap a length of DNA around itself in a positive sense. In previous work, the effect of ADPNP on the gyrase-DNA interaction has been assessed but has produced conflicting results; the aim of this work was to resolve this conflict. We have probed the wrapping of DNA around gyrase in the presence and in the absence of ADPNP using direct observation by atomic force microscopy (AFM). We confirm that gyrase indeed generates a significant curvature in DNA in the absence of nucleotide and we show that the addition of ADPNP leads to a complete loss of wrap. These results have been corroborated using a DNA relaxation assay involving topoisomerase I. We have re-analysed previous hydroxyl-radical footprinting and crystallography data, and highlight the fact that the gyrase-DNA complex is surprisingly asymmetric in the absence of nucleotide but is symmetric in the presence of ADPNP. We suggest a revised model for the conformation of DNA bound to the enzyme that is fully consistent with these AFM data, in which a closed loop of DNA is stabilised by the enzyme in the absence of ADPNP and is lost in the presence of nucleotide.     

The partition locus of plasmid pSC101 is a specific binding site for DNA gyrase.

A protein in extracts of Escherichia coli that specifically binds the stabilizing par sequence of pSC101 was identified as DNA gyrase. The purified enzyme protects par against digestion by DNase I and exonuclease III. Competition assays demonstrate that gyrase has a 40-fold higher affinity for the 100-bp par sequence than for nonspecific DNA and that par is the major gyrase-binding site in pSC101 derivatives used in this and other studies. Within par, AT-rich sequences occur with a pronounced 10-bp periodicity that is shifted by 5 bp from a similar periodicity of GC-rich sequences. As judged by DNase I digestion, the GC sequences are exposed on the outside of the DNA wrapped around gyrase. The data suggest that the site-specificity of DNA gyrase may be partly determined by the bendability of the DNA. A 4-bp deletion that interferes with Par function in vivo also reduces the affinity for gyrase in vitro. However, a deletion of par causes little reduction in superhelical density in vivo. We conclude that DNA gyrase, while involved in the Par function, may not affect plasmid stability through its supercoiling activity or by an influence on DNA replication.     

Site-specific cleavage of DNA by E. coli DNA gyrase.

E. coli DNA gyrase, which catalyzes the supercoiling of DNA, cleaves DNA site-specifically when oxolinic acid and sodium dodecylsulfate are added to the reaction. We studied the structure of the gyrasecleaved DNA because of its implications for the reaction mechanism and biological role of gyrase. Gyrase made a staggered cut, creating DNA termini with a free 3' hydroxyl and a 5' extension that provided a template primer for DNA polymerase. The cleaved DNA was resistant to labeling with T4 polynucleotide kinase even after treatment with proteinase K. Thus the denatured enzyme that remains attached to cleaved DNA is covalently bonded to both 5' terminal extensions. The 5' extensions of many gyrase cleavage fragments from phi X174, SV40 and Col E1 DNA were partially sequenced using repair with E. coli DNA polymerase I. No unique sequence existed within the cohesive ends, but G was the predominant first base incorporated by DNA polymerase I. The cohesive and sequences of four gyrase sites were determined, and they demonstrated a four base 5' extension. The dinucleotide TG, straddling the gyrase cut on one DNA strand, provided the only common bases within a 100 bp region surrounding the cleavage sites. Analysis of other cleavage fragments showed that cutting between a TG doublet is common to most, or all, gyrase cleavages. Other bases common to some of the sequenced sites were clustered nonrandomly around the TG doublet, and may be variable components of the cleavage sequence. This diverse recognition sequence with common elements is a pattern shared with several other specific nucleic acid-protein interactions.     

Identification of four GyrA residues involved in the DNA breakage-reunion reaction of DNA gyrase

DNA supercoiling by DNA gyrase involves the cleavage of a DNA helix, the passage of another helix through the break, and the religation of the first helix. The cleavage-religation reaction involves the formation of a 5'-phosphotyrosine intermediate with the GyrA subunit of the gyrase (A(2)B(2)) complex. We report the characterization of mutations near the active-site tyrosine residue in GyrA predicted to affect the cleavage-religation reaction of gyrase. We find that mutations at Arg32, Arg47, His78 and His80 inhibit DNA supercoiling and other reactions of gyrase. These effects are caused by the involvement of these residues in the DNA cleavage reaction; religation is largely unaffected by these mutations. We show that these residues cooperate with the active-site tyrosine residue on the opposite subunit of the GyrA dimer during the cleavage-religation reaction.     

Analysis of DNA cleavage by reverse gyrase from Sulfolobus shibatae B12.

Reverse gyrase is a type I-5' topoisomerase, which catalyzes a positive DNA supercoiling reaction in vitro. To ascertain how this reaction takes places, we looked at the DNA sequences recognized by reverse gyrase. We used linear DNA fragments of its preferred substrate, the viral SSV1 DNA, which has been shown to be positively supercoiled in vivo. The Sulfolobus shibatae B12 strain, an SSV1 virus host, was chosen for production of reverse gyrase. This naturally occurring system (SSV1 DNA-S. shibatae reverse gyrase) allowed us to determine which SSV1 DNA sequences are bound and cleaved by the enzyme with particularly high selectivity. We show that the presence of ATP decreases the number of cleaved complexes obtained whereas the non-hydrolyzable ATP analog adenosine 5'-[beta, gamma-imido]triphosphate increases it without changing the sequence specificity.     

Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193.     

Cre-lox recombination in Escherichia coli cells. Mechanistic differences from the in vitro reaction.

The mechanism of the Cre recombinase of bacteriophage P1 in Escherichia coli cells was analyzed by topological methods in order to determine the important features of the in vivo reaction. Lambda infection was used to introduce the cre gene into cells containing plasmid substrates. The products of Cre resolution on substrates with directly repeated sites were predominantly free circles, even though decatenation by DNA gyrase was blocked by the drug norfloxacin. Recombination by Cre was greatly stimulated by negative supercoiling, and inversion occurred inefficiently. These results are strikingly different from those found with purified enzyme in vitro. Our data imply that Cre recombination in vivo is much more tightly controlled than it is in vitro, and that Cre acts predominantly as a resolvase in vivo. We suggest a role for Cre-mediated recombination in P1 plasmid amplification that is consistent with the selectivity of the enzyme in vivo.     

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.     

Structural insights into quinolone antibiotic resistance mediated by pentapeptide repeat proteins: conserved surface loops direct the activity of a Qnr protein from a gram-negative bacterium

Quinolones inhibit bacterial type II DNA topoisomerases (e.g. DNA gyrase) and are among the most important antibiotics in current use. However, their efficacy is now being threatened by various plasmid-mediated resistance determinants. Of these, the pentapeptide repeat-containing (PRP) Qnr proteins are believed to act as DNA mimics and are particularly prevalent in gram-negative bacteria. Predicted Qnr-like proteins are also present in numerous environmental bacteria. Here, we demonstrate that one such, Aeromonas hydrophila AhQnr, is soluble, stable, and relieves quinolone inhibition of Escherichia coli DNA gyrase, thus providing an appropriate model system for gram-negative Qnr proteins. The AhQnr crystal structure, the first for any gram-negative Qnr, reveals two prominent loops (1 and 2) that project from the PRP structure. Deletion mutagenesis demonstrates that both contribute to protection of E. coli DNA gyrase from quinolones. Sequence comparisons indicate that these are likely to be present across the full range of gram-negative Qnr proteins. On this basis we present a model for the AhQnr:DNA gyrase interaction where loop1 interacts with the gyrase A 'tower' and loop2 with the gyrase B TOPRIM domains. We propose this to be a general mechanism directing the interactions of Qnr proteins with DNA gyrase in gram-negative bacteria.     

Enzymes as biosensors. 1. Enzyme memory and sensing chemical signals

When a free enzyme exists under different conformations that 'slowly' isomerize during the conversion of a substrate into a product, the corresponding 'slow' relaxation component may interfere with the steady-state component. The apparent steady-state rate that may be measured under these conditions is called the meta-steady-state rate for it refers to the existence of metastable states of the enzyme during the reaction. By contrast to the real steady-state rate, the meta-steady-state rate is dependent upon the initial state of the enzyme, that is on the respective concentrations of the free enzyme forms. The simplest model that may display this type of behaviour is the mnemonical model. For a fixed concentration of the last product of the reaction sequence the meta-steady state is different depending on that concentration being reached by an increase or a decrease of a previous concentration. This means that the meta-steady-state rate describes a hysteresis loop as the product concentration is increased and decreased. Owing to the existence of metastable states, the enzyme system behaves as a biosensor that is able to detect both a concentration and the direction of a concentration change. The existence of the hysteresis loop of the meta-steady-state rate implies that the two free enzyme forms display hysteresis as well. A chemical potential, called the sensing potential, is specifically associated with the 'perception' of the direction of the thermodynamic force generated by the decrease or the increase of the concentration of the ligand that binds to one of the enzyme conformations. The sensing potential of the enzyme conformer that does not bind the product increases and reaches a plateau as the chemical potential of that product is raised. Alternatively the sensing potential of the other conformer vanishes at low and high chemical potentials of the product and is significant for intermediate chemical potentials. Enzymes that display very slow conformation changes may thus be viewed as elementary sensor devices.     

ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings

Extracts of Drosophila embryos contain an enzymatic activity that converts circular DNAs into huge networks of catenated rings in an ATP-dependent fashion. The catenation activity is resolved into two protein components during purification. One component is a novel DNA topoisomerase that requires the presence of ATP in order to relax supercoiled DNA. We have shown that the ATP-dependent DNA topoisomerase relaxes DNA by a mechanism distinct from that of nicking-closing enzymes. The Drosophila ATP-dependent topoisomerase allows one segment of a circular DNA to pass through transient breaks in both strands at another site on the DNA circle without any relative rotation between the ends at the transient break. This mechanism can convert negative supertwists to positive twists and vice versa until a relaxed equilibrium state is reached. The formation of catenated rings is mediated by an analogous bimolecular reaction which can occur between two nonhomologous DNA circles. The catenation reaction is fully reversible: in the presence of the second protein component, circular DNA is converted quantitatively into catenated forms; in its absence, the ATP-dependent topoisomerase resolves catenated networks back into monomer circles. The Drosophila ATP-dependent topoisomerase appears to be closely related to E. coli DNA gyrase in that both use a similar mechanism to change the topology of DNA, both require ATP and both are inhibited by the antibiotic novobiocin. The presence of an enzyme that allows one DNA helix to pass freely through another could not only be useful in relaxation of topological constraints, but also may be involved in the folding and unfolding of eucaryotic chromosomes.     

The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication.

Mutants in bacterial topoisomerase (topo) IV are deficient in chromosomal partitioning. To investigate the basis of this phenotype, we examined plasmid DNA topology in conditionally lethal topo IV mutants. We found that dimeric catenated plasmids accumulated in vivo after topo IV inhibition. The catenanes were supercoiled, contained from 2 to > 32 nodes, and were the products of DNA synthesis. Electron microscopy and recombination tests proved that the catenanes have the unique structure predicted for replication intermediates. These data provide strong evidence for a model in which unlinking of the double helix can occur in two stages during DNA replication and for the critical role of topo IV in the second stage. The interlocks in the catenanes appear to be sequestered from DNA gyrase, perhaps by compartmentalization in an enzyme complex dedicated to partitioning.     

Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking.

Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling. Limited strand passage is allowed, but release of the transported DNA segment (T segment) via opening of the bottom dimer interface is prevented. In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked. These findings support a two-gate model for supercoiling by DNA gyrase and suggest that relaxation by gyrase is the reverse of supercoiling. Cross-linking a truncated version of gyrase (A64(2)B2), which lacks the DNA wrapping domains, does not block ATP-dependent relaxation. This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction. The mechanistic implications of these results are discussed.     

DNA Topoisomerase Inhibitors: Trapping a DNA-Cleaving Machine in Motion

Type II topoisomerases regulate DNA topology by making a double-stranded break in one DNA duplex, transporting another DNA segment through this break and then resealing it. Bacterial type IIA topoisomerase inhibitors, such as fluoroquinolones and novel bacterial topoisomerase inhibitors, can trap DNA cleavage complexes with double- or single-stranded cleaved DNA. To study the mode of action of such compounds, 21 crystal structures of a “gyrase^CORE” fusion truncate of Staphyloccocus aureus DNA gyrase complexed with DNA and diverse inhibitors have been published, as well as 4 structures lacking inhibitors. These structures have the DNA in various cleavage states and appear to track trajectories along the catalytic paths of the DNA cleavage/religation steps. The various conformations sampled by these multiple “gyrase^CORE” structures show rigid body movements of the catalytic GyrA WHD and GyrB TOPRIM domains across the dimer interface. Conformational changes common to all compound-bound structures suggest common mechanisms for DNA cleavage-stabilizing compounds. The structures suggest that S. aureus gyrase uses a single moving-metal ion for cleavage and that the central four base pairs need to be stretched between the two catalytic sites, in order for a scissile phosphate to attract a metal ion to the A-site to catalyze cleavage, after which it is “stored” in another coordination configuration (B-site) in the vicinity. We present a simplified model for the catalytic cycle in which capture of the transported DNA segment causes conformational changes in the ATPase domain that push the DNA gate open, resulting in stretching and cleaving the gate-DNA in two steps.