Widespread distribution of archaeal reverse gyrase in thermophilic bacteria suggests a complex history of vertical inheritance and lateral gene transfers

Reverse gyrase, an enzyme of uncertain funtion, is present in all hyperthermophilic archaea and bacteria. Previous phylogenetic studies have suggested that the gene for reverse gyrase has an archaeal origin and was transferred laterally (LGT) to the ancestors of the two bacterial hyperthermophilic phyla, Thermotogales and Aquificales. Here, we performed an in-depth analysis of the evolutionary history of reverse gyrase in light of genomic progress. We found genes coding for reverse gyrase in the genomes of several thermophilic bacteria that belong to phyla other than Aquificales and Thermotogales. Several of these bacteria are not, strictly speaking, hyperthermophiles because their reported optimal growth temperatures are below 80 degrees C. Furthermore, we detected a reverse gyrase gene in the sequence of the large plasmid of Thermus thermophilus strain HB8, suggesting a possible mechanism of transfer to the T. thermophilus strain HB8 involving plasmids and transposases. The archaeal part of the reverse gyrase tree is congruent with recent phylogenies of the archaeal domain based on ribosomal proteins or RNA polymerase subunits. Although poorly resolved, the complete reverse gyrase phylogeny suggests an ancient acquisition of the gene by bacteria via one or two LGT events, followed by its secondary distribution by LGT within bacteria. Finally, several genes of archaeal origin located in proximity to the reverse gyrase gene in bacterial genomes have bacterial homologues mostly in thermophiles or hyperthermophiles, raising the possibility that they were co-transferred with the reverse gyrase gene. Our new analysis of the reverse gyrase history strengthens the hypothesis that the acquisition of reverse gyrase may have been a crucial evolutionary step in the adaptation of bacteria to high-temperature environments. However, it also questions the role of this enzyme in thermophilic bacteria and the selective advantage its presence could provide.     

The linkage between reverse gyrase and hyperthermophiles: A review of their invariable association

With the discovery of reverse gyrase in 1972, from Yellowstone National Park, isolated from Sulfolobus acidocaldarius, it has been speculated as to why reverse gyrase can be found in all hyperthermophiles and just what exactly its role is in hyperthermophilic organisms. Hyperthermophiles have been defined as organisms with an optimal growth temperature of above 85°C. Reverse gyrase is responsible for the introduction of positive supercoils into closed circular DNA. This review of reverse gyrase in hyperthermophilic microorganisms summarizes the last two decades of research performed on hyperthermophiles and reverse gyrase in an effort to provide an up to date synopsis of their invariable association. From the data gathered for this review it is reasonable to hypothesize that reverse gyrase is closely tied to hyperthermophilic life.     

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.     

Reverse gyrase: an unusual DNA manipulator of hyperthermophilic organisms

Reverse gyrase is the only DNA topoisomerase capable of introducing positive supercoiling into DNA molecules. This unique activity reflects a distinctive arrangement of the protein, which is composed of a topoisomerase IA module fused to a domain containing sequence motives typical of helicases; however, reverse gyrase works neither like a canonical topoisomerase IA nor like a helicase. Extensive genomic analysis has shown that reverse gyrase is present in all organisms living above 70 degrees C and in some of those living at 60- 70 degrees C, but is invariably absent in organisms living at mesophilic temperatures. For its peculiar distribution and biochemical activity, the enzyme has been suggested to play a role in maintenance of genome stability at high temperature. We review here recent phylogenetic, biochemical and structural data on reverse gyrase and discuss the possible role of this enzyme in the biology of hyperthermophilic organisms.     

Speculations on the origin of life and thermophily: Review of available information on reverse gyrase suggests that hyperthermophilic procaryotes are not so primitive

All present-day hyperthermophiles studied so far (eitherBacteria orArchaea) contain a unique DNA topoisomerase, reverse gyrase, which probably helps to stabilize genomic DNA at high temperature. Herein the data relating this enzyme is reviewed and discussed from the perspective of the nature of the last detectable common ancestor and the origin of life. The sequence of the gene encoding reverse gyrase from an archaeon,Sulfolobus acidocaldarius, suggests that this enzyme contains both a helicase and a topoisomerase domains (Confalonieriet al.,Proc. Natl. Acad. Sci., 1993, 90, 4735). Accordingly, it has been proposed that reverse gyrase originated by the fusion of DNA helicase and DNA topoisomerase genes. If reverse gyrase is essential for life at high temperature, its composite structure suggests that DNA helicases and topoisomerases appeared independently and first evolved in a mesophilic world. Such scenario contradicts the hypothesis that a direct link connects present day hyperthermophiles to a hot origin of life. We discuss different patterns for the early cellular evolution in which reverse gyrase appeared either before the emergence of the last common ancestor ofArchaea, Bacteria andEucarya, or in a lineage common to the two procaryotic domains. The latter scenario could explain why all today hyperthermophiles are procaryotes.     

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.     

Reverse gyrase is not a prerequisite for hyperthermophilic life

We disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain. We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures. However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90 degrees C. This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life.     

Reverse gyrase functions as a DNA renaturase: annealing of complementary single-stranded circles and positive supercoiling of a bubble substrate

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.     

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.     

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.     

Distribution of reverse gyrase in representative species of eubacteria and archaebacteria

Reverse gyrase is a topoisomerase which positively supercoils closed circular plasmid DNA. Reverse gyrase activity is restricted to the thermoacidophilic group of archaebacteria. Thermophilic methanogens and eubacteria and all mesophilic organisms screened had no reverse gyrase activity. The result supports the deep phylogenetic divergence in archaebacterial evolution.     

Reverse gyrase, a hallmark of the hyperthermophilic archaebacteria.

Investigation of the presence of a reverse gyrase-like activity in archaebacteria revealed wide distribution of this activity in hyperthermophilic species, including methanogens and sulfur-dependent organisms. In contrast, no reverse gyrase activity was detected in mesophilic and moderately thermophilic organisms, which exhibited only an ATP-independent activity of DNA relaxation. These results suggest that the presence of reverse gyrase in archaebacteria is tightly linked to the high growth temperatures of these organisms. With respect to antigenic properties, the enzyme appeared similar among members of the genus Sulfolobus. In contrast, no close antigenic relatedness was found between the reverse gyrase of members of the order Sulfolobales and that of the other hyperthermophilic organisms.     

Reverse gyrase: an insight into the role of DNA-topoisomerases

Reverse gyrase was discovered more than twenty years ago. Recent biochemical and structural results have greatly enhanced our understanding of their positive supercoiling mechanism. In addition to new biochemical properties, a fine tuning of reverse gyrase regulation in response to DNA damaging agents has been recently described. These data give us a new insight in the cellular role of reverse gyrase. Moreover, it has been proposed that reverse gyrase has been implicated in genome stability.     

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 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.     

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.     

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.     

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.     

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.