The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.     

Reprint of “The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage”

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.     

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.     

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.     

Helicase-appended Topoisomerases: New Insight into the Mechanism of Directional Strand Transfer

DNA strand passage through an enzyme-mediated gate is a key step in the catalytic cycle of topoisomerases to produce topological transformations in DNA. In most of the reactions catalyzed by topoisomerases, strand passage is not directional; thus, the enzyme simply provides a transient DNA gate through which DNA transport is allowed and thereby resolves the topological entanglement. When studied in isolation, the type IA topoisomerase family appears to conform to this rule. Interestingly, type IA enzymes can carry out directional strand transport as well. We examined here the biochemical mechanism for directional strand passage of two type IA topoisomerases: reverse gyrase and a protein complex of topoisomerase IIIα and Bloom helicase. These enzymes are able to generate vectorial strand transport independent of the supercoiling energy stored in the DNA molecule. Reverse gyrase is able to anneal single strands, thereby increasing linkage number of a DNA molecule. However, topoisomerase IIIα and Bloom helicase can dissolve DNA conjoined with a double Holliday junction, thus reducing DNA linkage. We propose here that the helicase or helicase-like component plays a determinant role in the directionality of strand transport. There is thus a common biochemical ground for the directional strand passage for the type IA topoisomerases.     

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.     

Guiding strand passage: DNA-induced movement of the gyrase C-terminal domains defines an early step in the supercoiling cycle

DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA in a strand passage mechanism. A double-stranded segment of DNA, the T-segment, is passed through the gap in a transiently cleaved G-segment by coordinated closing and opening of three protein interfaces in gyrase. T-segment capture is thought to be guided by the C-terminal domains of the GyrA subunit of gyrase that wrap DNA around their perimeter and cause a DNA-crossing with a positive handedness. We show here that the C-terminal domains are in a downward-facing orientation in the absence of DNA, but swing up and rotate away from the gyrase body when DNA binds. The upward movement of the C-terminal domains is an early event in the catalytic cycle of gyrase that is triggered by binding of a G-segment, and first contacts of the DNA with the C-terminal domains, and contributes to T-segment capture and subsequent strand passage.     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Potassium ions are required for nucleotide-induced closure of gyrase N-gate

DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA by a strand passage mechanism that requires coordinated opening and closing of three protein interfaces, the N-, DNA-, and C-gates. ATP binding to the GyrB subunits of gyrase causes dimerization and N-gate closure. The closure of the N-gate is a key step in the gyrase catalytic cycle, as it captures the DNA segment to be transported and poises gyrase toward strand passage. We show here that K(+) ions are required for DNA supercoiling but are dispensable for ATP-independent DNA relaxation. Although DNA binding, distortion, wrapping, and DNA-induced narrowing of the N-gate occur in the absence of K(+), nucleotide-induced N-gate closure depends on their presence. Our results provide evidence that K(+) ions relay small conformational changes in the nucleotide-binding pocket to the formation of a tight dimer interface at the N-gate by connecting regions from both GyrB monomers and suggest an important role for K(+) in synchronization of N-gate closure and DNA-gate opening.     

The Acidic C-terminal Tail of the GyrA Subunit Moderates the DNA Supercoiling Activity of Bacillus subtilis Gyrase

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.     

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.     

A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism

DNA gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or gyrase-DNA interactions. More importantly, the ternary complex of gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of gyrase inhibitors. DNA gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing gyrase mechanism.     

Mechanism of the reaction catalyzed by the DNA untwisting enzyme: attachment of the enzyme to 3'-terminus of the nicked DNA.

The rat liver DNA untwisting enzyme introduces a transient nick into duplex DNA. The enzyme has been shown to be covalently attached to one of the ends of the broken strand in the nicked intermediate (Champoux, 1977). The broken strand containing bound enzyme is shown to be susceptible to phosphorylation by polynucleotide kinase. Therefore, the DNA untwisting enzyme must be attached to the strand at the 3′-phosphate terminus, and this linkage probably conserves the energy required for resealing the single-strand break.     

Crystal structures of Thermotoga maritima reverse gyrase: inferences for the mechanism of positive DNA supercoiling

Reverse gyrase is an ATP-dependent topoisomerase that is unique to hyperthermophilic archaea and eubacteria. The only reverse gyrase structure determined to date has revealed the arrangement of the N-terminal helicase domain and the C-terminal topoisomerase domain that intimately cooperate to generate the unique function of positive DNA supercoiling. Although the structure has elicited hypotheses as to how supercoiling may be achieved, it lacks structural elements important for supercoiling and the molecular mechanism of positive supercoiling is still not clear. We present five structures of authentic Thermotoga maritima reverse gyrase that reveal a first view of two interacting zinc fingers that are crucial for positive DNA supercoiling. The so-called latch domain, which connects the helicase and the topoisomerase domains is required for their functional cooperation and presents a novel fold. Structural comparison defines mobile regions in parts of the helicase domain, including a helical insert and the latch that are likely important for DNA binding during catalysis. We show that the latch, the helical insert and the zinc fingers contribute to the binding of DNA to reverse gyrase and are uniquely placed within the reverse gyrase structure to bind and guide DNA during strand passage. A possible mechanism for positive supercoiling by reverse gyrases is presented.     

A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

DNA gyrase is the only topoisomerase able to introduce negative supercoils into DNA. Absent in humans, gyrase is a successful target for antibacterial drugs. However, increasing drug resistance is a serious problem and new agents are urgently needed. The naturally-produced Escherichia coli toxin CcdB has been shown to target gyrase by what is predicted to be a novel mechanism. CcdB has been previously shown to stabilize the gyrase ‘cleavage complex’, but it has not been shown to inhibit the catalytic reactions of gyrase. We present data showing that CcdB does indeed inhibit the catalytic reactions of gyrase by stabilization of the cleavage complex and that the GyrA C-terminal DNA-wrapping domain and the GyrB N-terminal ATPase domain are dispensable for CcdB's action. We further investigate the role of specific GyrA residues in the action of CcdB by site-directed mutagenesis; these data corroborate a model for CcdB action based on a recent crystal structure of a CcdB–GyrA fragment complex. From this work, we are now able to present a model for CcdB action that explains all previous observations relating to CcdB–gyrase interaction. CcdB action requires a conformation of gyrase that is only revealed when DNA strand passage is taking place.     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate.