Investigating the role of the latch in the positive supercoiling mechanism of reverse gyrase

Reverse gyrase is the only topoisomerase known to positively supercoil DNA and the only protein unique to hyperthermophiles. The enzyme comprises an N-terminal ATPase domain and a C-terminal topoisomerase I domain, which interact to couple the hydrolysis of ATP to the overwinding of DNA. The part of the ATPase domain termed the "latch" represses topoisomerase activity in the absence of nucleotide. Here I provide evidence that the latch, in addition to its regulatory role, participates in the supercoiling mechanism during the DNA cleavage and religation steps. The latch also contributes to the coordination of ATP hydrolysis and positive supercoiling by inhibiting ATPase activity in the absence of supercoiling. The latch therefore plays an important role in the communication between the two domains of reverse gyrase.     

The reverse gyrase helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain

Reverse gyrase is a topoisomerase that introduces positive supercoils into DNA in an ATP-dependent manner. It is unique to hyperthermophilic archaea and eubacteria, and has been proposed to protect their DNA from damage at high temperatures. Cooperation between its N-terminal helicase-like and the C-terminal topoisomerase domain is required for positive supercoiling, but the precise role of the helicase-like domain is currently unknown. Here, the characterization of the isolated helicase-like domain from Thermotoga maritima reverse gyrase is presented. We show that the helicase-like domain contains all determinants for nucleotide binding and ATP hydrolysis. Its intrinsic ATP hydrolysis is significantly stimulated by ssDNA, dsDNA and plasmid DNA. During the nucleotide cycle, the helicase-like domain switches between high- and low-affinity states for dsDNA, while its affinity for ssDNA in the ATP and ADP states is similar. In the context of reverse gyrase, the differences in DNA affinities of the nucleotide states are smaller, and the DNA-stimulated ATPase activity is strongly reduced. This inhibitory effect of the topoisomerase domain decelerates the progression of reverse gyrase through the nucleotide cycle, possibly providing optimal coordination of ATP hydrolysis with the complex reaction of DNA supercoiling.     

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.     

The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

Reverse gyrase is the only topoisomerase that can introduce positive supercoils into DNA in an ATP-dependent process. It has a modular structure and harnesses a helicase-like domain to support a topoisomerase activity, thereby creating the unique function of positive DNA supercoiling. The isolated topoisomerase domain can relax negatively supercoiled DNA, an activity that is suppressed in reverse gyrase. The isolated helicase-like domain is a nucleotide-dependent switch that is attenuated by the topoisomerase domain. Inter-domain communication thus appears central for the functional cooperation of the two domains. The latch, an insertion into the helicase-like domain, has been suggested as an important element in coordinating their activities. Here, we have dissected the influence of the latch on nucleotide and DNA binding to the helicase-like domain, and on DNA supercoiling by reverse gyrase. We find that the latch is required for positive DNA supercoiling. It is crucial for the cooperativity of DNA and nucleotide binding to the helicase-like domain. The latch contributes to DNA binding, and affects the preference of reverse gyrase for ssDNA. Thus, the latch coordinates the individual domain activities by modulating the helicase-like domain, and by communicating changes in the nucleotide state to the topoisomerase domain.     

Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling

Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.     

The Reverse Gyrase from Pyrobaculum calidifontis,a Novel Extremely Thermophilic DNA Topoisomerase Endowed with DNA Unwinding and Annealing Activities

Reverse gyrase is a DNA topoisomerase specific for hyperthermophilic bacteria and archaea. It catalyzes the peculiar ATP-dependent DNA-positive supercoiling reaction and might be involved in the physiological adaptation to high growth temperature. Reverse gyrase comprises an N-terminal ATPase and a C-terminal topoisomerase domain, which cooperate in enzyme activity, but details of its mechanism of action are still not clear. We present here a functional characterization of PcalRG, a novel reverse gyrase from the archaeon Pyrobaculum calidifontis. PcalRG is the most robust and processive reverse gyrase known to date; it is active over a wide range of conditions, including temperature, ionic strength, and ATP concentration. Moreover, it holds a strong ATP-inhibited DNA cleavage activity. Most important, PcalRG is able to induce ATP-dependent unwinding of synthetic Holliday junctions and ATP-stimulated annealing of unconstrained single-stranded oligonucleotides. Combined DNA unwinding and annealing activities are typical of certain helicases, but until now were shown for no other reverse gyrase. Our results suggest for the first time that a reverse gyrase shares not only structural but also functional features with evolutionary conserved helicase-topoisomerase complexes involved in genome stability.     

Intrinsic DNA-dependent ATPase activity of reverse gyrase

Reverse gyrase is a type I DNA topoisomerase that promotes positive supercoiling of closed-circular double-stranded DNA through an ATP-dependent reaction, and it was purified from an archaebacterium, Sulfolobus. When ATP is replaced by UTP, GTP, or CTP, this enzyme just relaxes the negatively supercoiled closed-circular double-stranded DNA. We found that reverse gyrase hydrolyzes ATP through a double-stranded DNA-dependent reaction. The superhelicity of the DNA did not affect the ATPase activity. However, reverse gyrase does not hydrolyze UTP, GTP, or CTP. Therefore, any of the four nucleotide 5'-triphosphates acts as an effector for the topoisomerase activity of reverse gyrase, but only ATP supports the positive supercoiling of closed-circular double-stranded DNA, through the energy released on its hydrolysis. Single-stranded DNA was a much more potent cofactor for the ATPase activity of the enzyme than double-stranded DNA, and it acted as a potent inhibitor for the topoisomerase activity on double-stranded DNA. These results indicate that reverse gyrase has higher affinity to single-stranded DNA than to double-stranded DNA, which suggests a cellular function of the enzyme.     

Adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) promotes positive supercoiling of DNA by T. maritima reverse gyrase

Reverse gyrases are topoisomerases that catalyze ATP-dependent positive supercoiling of circular covalently closed DNA. They consist of an N-terminal helicase-like domain, fused to a C-terminal topoisomerase I-like domain. Most of our knowledge on reverse gyrase-mediated positive DNA supercoiling is based on studies of archaeal enzymes. To identify general and individual properties of reverse gyrases, we set out to characterize the reverse gyrase from a hyperthermophilic eubacterium. Thermotoga maritima reverse gyrase relaxes negatively supercoiled DNA in the presence of ADP or the non-hydrolyzable ATP-analog ADPNP. Nucleotide binding is necessary, but not sufficient for the relaxation reaction. In the presence of ATP, positive supercoils are introduced at temperatures above 50 degrees C. However, ATP hydrolysis is stimulated by DNA already at 37 degrees C, suggesting that reverse gyrase is not frozen at this temperature, but capable of undergoing inter-domain communication. Positive supercoiling by reverse gyrase is strictly coupled to ATP hydrolysis. At the physiological temperature of 75 degrees C, reverse gyrase binds and hydrolyzes ATPgammaS. Surprisingly, ATPgammaS hydrolysis is stimulated by DNA, and efficiently promotes positive DNA supercoiling, demonstrating that inter-domain communication during positive supercoiling is fully functional with both ATP and ATPgammaS. These findings support a model for communication between helicase-like and topoisomerase domains in reverse gyrase, in which an ATP and DNA-induced closure of the cleft in the helicase-like domain initiates a cycle of conformational changes that leads to positive DNA supercoiling.     

Reverse Gyrase Transiently Unwinds Double-Stranded DNA in an ATP-Dependent Reaction

Reverse gyrase is a unique DNA topoisomerase that catalyzes the introduction of positive supercoils into DNA in an ATP-dependent reaction. It consists of a helicase domain that functionally cooperates with a topoisomerase domain. Different models for the catalytic mechanism of reverse gyrase that predict a central role of the helicase domain have been put forward. The helicase domain acts as a nucleotide-dependent conformational switch that alternates between open and closed states with different affinities for single- and double-stranded DNA. It has been suggested that the helicase domain can unwind double-stranded regions, but helicase activity has not been demonstrated as yet. Here, we show that the isolated helicase domain and full-length reverse gyrase can transiently unwind double-stranded regions in an ATP-dependent reaction. The latch region of reverse gyrase, an insertion into the helicase domain, is required for DNA supercoiling. Strikingly, the helicase domain lacking the latch cannot unwind DNA, linking unwinding to DNA supercoiling. The unwinding activity may provide and stabilize the single-stranded regions required for strand passage by the topoisomerase domain, either de novo or by expanding already existing unpaired regions that may form at high temperatures.     

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.     

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.     

Mutational analysis of the helicase-like domain of Thermotoga maritima reverse gyrase

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.     

Studies of a positive supercoiling machine. Nucleotide hydrolysis and a multifunctional "latch" in the mechanism of reverse gyrase

Reverse gyrase, the only topoisomerase known to positively supercoil DNA, has an N-terminal ATPase domain that drives the activity of a topoisomerase domain. This study shows that the N-terminal domain represses topoisomerase activity in the absence of nucleotide, and nucleotide binding is sufficient to relieve the repression. A "latch" region in the N-terminal part was observed to close over the topoisomerase domain in the reverse gyrase crystal structure. Mutants lacking all or part of the latch relax DNA in the absence of nucleotide, indicating that this region mediates topoisomerase repression. The mutants also show altered DNA-dependent ATPase activity, suggesting that the latch may be involved in coupling nucleotide hydrolysis to supercoiling. It is not required for this process, however, because the mutants can still positively supercoil DNA. Nucleotide hydrolysis is essential to the specificity of reverse gyrase for increasing the linking number of DNA. Although with ATP the enzyme performs strand passage always toward increasing linking number, it can increase or decrease the linking number in the presence of a nonhydrolyzable ATP analog. This suggests that the mechanism of reverse gyrase is best described by a combination of recently proposed models.     

The conformational flexibility of the helicase-like domain from Thermotoga maritima reverse gyrase is restricted by the topoisomerase domain

Reverse gyrase is the only enzyme known to introduce positive supercoils into DNA. Positive supercoiling is achieved by the functional cooperation of a helicase-like and a topoisomerase domain. The isolated helicase-like domain is a DNA-stimulated ATPase, and the isolated topoisomerase domain can relax supercoiled DNA. In the context of reverse gyrase, these individual activities are suppressed or attenuated. The helicase-like domain of Thermotoga maritima reverse gyrase is a nucleotide-dependent conformational switch that binds DNA and ATP cooperatively. It provides a nucleotide-dependent DNA-binding site to reverse gyrase and thus serves as a valuable model for the investigation of the effect of nucleotides on DNA processing by reverse gyrase that is key to its supercoiling activity. To improve our understanding of the structural basis for the functional cooperation of a helicase domain with a DNA topoisomerase, we have determined the structures of the isolated helicase-like domain of T. maritima reverse gyrase in five different conformations. Comparison of these structures reveals extensive domain flexibility in the absence of conformational restrictions by the topoisomerase that is consistent with single-molecule Fo?rster resonance energy transfer experiments presented here. The structure of the first ADP-bound form provides novel details about nucleotide binding to reverse gyrase. It demonstrates that reverse gyrases use the canonical nucleotide binding mode common to superfamily 2 helicases despite large deviations in the conserved motifs. A characteristic insert region adopts drastically different structures in different reverse gyrases. Counterparts of this insert region are located at very different positions in other DNA-processing enzymes but may point toward a general role in DNA strand separation.     

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.     

What makes a type IIA topoisomerase a gyrase or a Topo IV?

Type IIA topoisomerases catalyze a variety of different reactions: eukaryotic topoisomerase II relaxes DNA in an ATP-dependent reaction, whereas the bacterial representatives gyrase and topoisomerase IV (Topo IV) preferentially introduce negative supercoils into DNA (gyrase) or decatenate DNA (Topo IV). Gyrase and Topo IV perform separate, dedicated tasks during replication: gyrase removes positive supercoils in front, Topo IV removes pre-catenanes behind the replication fork. Despite their well-separated cellular functions, gyrase and Topo IV have an overlapping activity spectrum: gyrase is also able to catalyze DNA decatenation, although less efficiently than Topo IV. The balance between supercoiling and decatenation activities is different for gyrases from different organisms. Both enzymes consist of a conserved topoisomerase core and structurally divergent C-terminal domains (CTDs). Deletion of the entire CTD, mutation of a conserved motif and even by just a single point mutation within the CTD converts gyrase into a Topo IV-like enzyme, implicating the CTDs as the major determinant for function. Here, we summarize the structural and mechanistic features that make a type IIA topoisomerase a gyrase or a Topo IV, and discuss the implications for type IIA topoisomerase evolution.     

The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea

Abstract: Hyperthermophilic archaea exhibit a unique pattern of DNA topoisomerase activities. They have a peculiar enzyme, reverse gyrase, which introduces positive superturns into DNA at the expense of ATP. This enzyme has been found in all hyperthermophiles tested so far (including Bacteria) but never in mesophiles. Reverse gyrases are formed by the association of a helicase-like domain and a 5'-type I DNA topoisomerase. These two domains might be located on the same polypeptide. However, in the methanogenic archaeon Methanopyrus kandleri , the topoisomerase domain is divided between two subunits. Besides reverse gyrase, Archaea contain other type I DNA topoisomerases; in particular, M. kandleri harbors the only known procaryotic 3'-type I DNA topoisomerase (Topo V). Hyperthermophilic archaea also exhibit specific type II DNA topoisomerases (Topo II), i.e. whereas mesophilic Bacteria have a Topo II that produces negative supercoiling (DNA gyrase), the Topo II from Sulfolobus and Pyrococcus lack gyrase activity and are the smallest enzymes of this type known so far. This peculiar pattern of DNA topoisomerases in hyperthermophilic archaea is paralleled by a unique DNA topology, i.e. whereas DNA isolated from Bacteria and Eucarya is negatively supercoiled, plasmidic DNA from hyperthermophilic archaea are from relaxed to positively supercoiled. The possible evolutionary implications of these findings are discussed in this review. We speculate that gyrase activity in mesophiles and reverse gyrase activity in hyperthermophiles might have originated in the course of procaryote evolution to balance the effect of temperature changes on DNA structure.     

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.     

Reverse gyrase—recent advances and current mechanistic understanding of positive DNA supercoiling

Reverse gyrases are topoisomerases that introduce positive supercoils into DNA in an ATP-dependent reaction. They consist of a helicase domain and a topoisomerase domain that closely cooperate in catalysis. The mechanism of the functional cooperation of these domains has remained elusive. Recent studies have shown that the helicase domain is a nucleotide-regulated conformational switch that alternates between an open conformation with a low affinity for double-stranded DNA, and a closed state with a high double-stranded DNA affinity. The conformational cycle leads to transient separation of DNA duplexes by the helicase domain. Reverse gyrase-specific insertions in the helicase module are involved in binding to single-stranded DNA regions, DNA unwinding and supercoiling. Biochemical and structural data suggest that DNA processing by reverse gyrase is not based on sequential action of the helicase and topoisomerase domains, but rather the result of an intricate cooperation of both domains at all stages of the reaction. This review summarizes the recent advances of our understanding of the reverse gyrase mechanism. We put forward and discuss a refined, yet simple model in which reverse gyrase directs strand passage toward increasing linking numbers and positive supercoiling by controlling the conformation of a bound DNA bubble.     

Separate and combined biochemical activities of the subunits of a naturally split reverse gyrase

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single- and double-stranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.