Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling
2000; Elsevier BV; Volume: 275; Issue: 26 Linguagem: Inglês
10.1074/jbc.m910091199
ISSN1083-351X
AutoresAnne‐Cécile Déclais, Janine Marsault, Fabrice Confalonieri, Claire Bouthier de la Tour, Michel Duguet,
Tópico(s)Reproductive tract infections research
ResumoReverse 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 inEscherichia 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 °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. 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 inEscherichia 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 °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. glutathioneS-transferase dithiothreitol 5′-adenylyl-β,γ-imidodiphosphate Reverse gyrase is one of the most typical examples of the originality of biological macromolecules produced by hyperthermophilic organisms. Initially discovered in an Archaeon (see Refs. 1.Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (193) Google Scholar and 2.Duguet M. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. Springer-Verlag, Heidelberg, Germany1995: 85-114Google Scholar for a review), it has been further isolated from a variety of archaea and bacteria and has been recognized as a marker of hyperthermophiles, independently of the domain of life to which it belongs (3.Collin R.G. Morgan H.W. Musgrave D.R. Daniel R.M. FEMS Microbiol. Lett. 1988; 55: 235-240Crossref Scopus (22) Google Scholar, 4.Andera L. Mikulik K. Savelyeva N.D. FEMS Microbiol. Lett. 1993; 110: 107-112Crossref Scopus (0) Google Scholar, 5.Bouthier de La Tour C. Portemer C. Kaltoum H. Duguet M. J. Bacteriol. 1998; 180: 274-281Crossref PubMed Google Scholar). The basic activity performed in vitro by this enzyme is the production of positive supercoils in a closed circular DNA, a thermodynamically unfavored reaction that is driven by ATP. This activity provided possible explanations for the specific presence of reverse gyrase in hyperthermophiles, since positive supercoiling may stabilize the double helix at high temperatures, preventing local opening of the helix or allowing duplex "renaturation" after the passage of a transcription complex (6.Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google Scholar). An additional "raison d'être" of reverse gyrase could be the use of positive supercoiling to assemble chromatin-like structures in some hyperthermophiles (7.Forterre P. Charbonnier F. Marguet E. Harper F. Henkes G. Biochem. Soc. Symp. 1992; 58: 99-112PubMed Google Scholar). More recently, a similar activity was proposed to be present in eukaryotes (8.Gangloff S. McDonald J.-P. Bendixen C. Arthur L. Rothstein R. Mol. Cell. Biol. 1994; 14: 8391-8398Crossref PubMed Scopus (615) Google Scholar), where it could be involved in disrupting nucleosomal structures or in destabilizing illegitimate recombination intermediates (9.Duguet M. J. Cell Sci. 1997; 110: 1345-1350Crossref PubMed Google Scholar), thus giving reverse gyrase a more universal status. A crucial question that could shed some light on the function of reverse gyrase remains unanswered. What is the mechanism by which this sophisticated enzyme is able to catalyze a positive supercoiling reaction? Several years ago, two unexpected results somewhat clarified the problem. One was the finding that reverse gyrase, despite its unique ATP dependence, was a type I topoisomerase, transiently bound to the 5′ end of the cleaved strand (10.Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (39) Google Scholar), as the other members of the type I-5′ family (11.Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Crossref PubMed Scopus (2054) Google Scholar). The second was that stoichiometric binding of reverse gyrase to a form II DNA in the absence of ATP decreased the linking number of DNA after closure by a ligase, a property that was interpreted as DNA unwinding and was reminiscent of helicases (10.Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (39) Google Scholar). An additional clue to the reverse gyrase mechanism was later provided by the cloning and sequencing of the enzyme from Sulfolobus acidocaldarius in our laboratory (12.Confalonieri F. Elie C. Nadal M. de La Tour C.B. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar). The sequence that came out was clearly divided into two halves. The N-terminal half exhibited the usual signatures of a family of DNA/RNA helicases, and the C-terminal half was similar to the type I-5′ topoisomerases, confirming the previous results. All of the other recently sequenced reverse gyrase genes also contain the signatures of these two protein families, although the gene structure can be somewhat different (13.Krah R. O'Dea M.H. Gellert M. J. Biol. Chem. 1997; 272: 13986-13990Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Based on this structure in two domains, we proposed a "dynamic" model for the mechanism in which we assumed that the N-terminal domain had an ATP-dependent helicase activity; helix tracking by this domain was supposed to produce a wave of positive supercoiling ahead of reverse gyrase and of negative supercoiling behind (14.Liu L.F. Wang J.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7024-7027Crossref PubMed Scopus (1472) Google Scholar). Specific relaxation of these negative supercoils by the topoisomerase I domain was supposed to finally produce net positive supercoiling (12.Confalonieri F. Elie C. Nadal M. de La Tour C.B. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar). This model supported the idea that in eukaryotes the function of reverse gyrase could be fulfilled by the association of a helicase and a topoisomerase (9.Duguet M. J. Cell Sci. 1997; 110: 1345-1350Crossref PubMed Google Scholar). However, recent results (15.Kovalsky O. Kozyavkin S.A. Slesarev A.I. Nucleic Acids Res. 1990; 18: 2801-2805Crossref PubMed Scopus (30) Google Scholar, 16.Jaxel C. Duguet M. Nadal M. Eur. J. Biochem. 1999; 260: 103-111Crossref PubMed Scopus (21) Google Scholar) on the sequence specificity of reverse gyrase are difficult to reconcile with the above model, which implies that the protein moves rapidly along the DNA. Moreover, the model was exclusively based on protein sequence comparisons, so that the precise mechanism of reverse gyrase is still a matter of hypotheses. In order to confirm these, we expressed separately in Escherichia coli the entire 1247 amino acid putative coding sequence ofS. acidocaldarius reverse gyrase and its putative helicase-like (amino acids 1–612) and topoisomerase (residues 610–1247) domains. In the present paper, we show that full-length recombinant reverse gyrase, when expressed at 37 °C in E. coli, is able to sustain efficient positive supercoiling at 75 °C in the presence of ATP, without requiring any preheating treatment. As expected, mutation of the putative active site tyrosine 964 into a phenylalanine completely abolished this activity. In addition, the putative topoisomerase I domain alone, expressed in E. coli, exhibited an ATP-independent DNA relaxation activity on negative supercoils at 75 °C. By contrast, we failed to detect any helicase activity for the N-terminal domain as for the whole reverse gyrase. However, simple mixing of the two domains reconstitutes the ATP-dependent positive supercoiling typical of reverse gyrase. The interaction between the domains seems rather specific, since the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like domain cannot be replaced by a thermophilic helicase. Finally, we show that the stoichiometric DNA unwinding activity is not an intrinsic property of the helicase-like domain, contrary to our previous estimation (17.Jaxel C. Bouthier de La Tour C. Duguet M. Nadal M. Nucleic Acids Res. 1996; 24: 4668-4675Crossref PubMed Scopus (28) Google Scholar). Like the supercoiling activity, DNA unwinding requires the presence of both domains, be they separately expressed or linked in a single polypeptide. These results suggest a mechanism in which the N-terminal domain does not directly unwind DNA but is more likely a protein motor, driving conformational changes within the whole enzyme. Bacterial strains used in this work were DH5α (cloning) and XL mutS Kanr (mutagenesis). Culture media were from Life Technologies, Inc. Restriction enzymes, T4, and Thermus DNA ligases were purchased from New England Biolabs, and oligonucleotides were from Genosys. Reverse gyrase and its putative domains were produced in E. coli as glutathioneS-transferase (GST)1 fusion proteins, with the pGEX expression system described by Smith and Johnson (18.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5026) Google Scholar). The reverse gyrase complete coding sequence from S. acidocaldarius was inserted in two steps into the pGEX-2TH vector. (i) We used polymerase chain reaction to insert a BamHI site immediately 5′ to the first codon and to amplify the 5′ terminus of the gene from a recombinant λ phage carrying the entire gene (12.Confalonieri F. Elie C. Nadal M. de La Tour C.B. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar). The amplified fragment was digested by BamHI andBsaBI. (ii) The same λ phage was digested byBsaBI and HindIII to prepare the 3′ part of the gene, and both fragments were inserted into theBamHI-HindIII site of the vector. The design of the reverse gyrase domains and their boundaries was exclusively based on the sequence: we used secondary structure predictions (Mac Molly package, Soft Gene, GmbH) to define a flexible loop, located about 15 amino acids upstream the first topoisomerase motif, as the junction between the two putative domains. We thus defined the helicase-like domain as amino acids 1–612 and the topoisomerase domain as amino acids 610–1247. The corresponding DNA segments were inserted into the cloning vector by using the same strategy as for the complete enzyme. Mutagenesis of the putative active site tyrosine into a phenylalanine was performed with the ChameleonTM kit of Stratagene, as described by the manufacturer, to yield the RG-Y964F and RG Top Y352F mutants. Two liters of LB broth were inoculated with E. coli DH5α strain carrying the various plasmid constructions. Cells were grown at 37 °C with aeration to an A 600 of 0.8, and isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 0.1 mm. After 2 h, cells were quickly cooled on ice and harvested. The cell paste (4 g) was resuspended in 10 volumes of lysis buffer (50 mm Tris, pH 7.4, 100 mm NaCl, 1 mm EDTA, 1 mmEGTA, 10% glycerol, 1% Nonidet P-40), and the cells were lysed in a French press at 9,000 pounds/square inch. Cellular debris were removed by centrifugation at 10,000 × g for 15 min, and the fusion proteins were purified on glutathione-agarose (from Sigma) using a batch procedure modified from Smith and Johnson (18.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5026) Google Scholar); the crude extract was stirred with 1.6 ml of glutathione-agarose for 1 h at 4 °C, and the resin was harvested by centrifugation at 500 ×g for 10 min. After extensive washing with 1 mNaCl in 50 mm Tris, pH 8.0, the fusion proteins were eluted by 20 mm reduced glutathione. The GST tag was removed by incubation for 3 h at 26 °C with thrombin. In the case of the full-length reverse gyrase, an additional purification step on phenyl-Sepharose was necessary to remove fragments produced by proteolysis or incomplete expression. The sample, adjusted to 300 mm NaCl, was loaded onto a 0.5-ml phenyl-Sepharose CL-4B column (Amersham Pharmacia Biotech) equilibrated in buffer A (50 mmNa2HPO4/NaH2PO4, pH 7.0, 1 mm DTT, 1 mm EDTA, 300 mmNaCl). After the column was washed by 25% ethylene glycol in the same buffer, reverse gyrase was eluted by 60% ethylene glycol. The various fractions were analyzed by SDS-polyacrylamide gel electrophoresis and checked for topoisomerase activities. This assay was essentially based on the displacement of a labeled oligonucleotide from a partial duplex, with modifications for an adaptation to high temperatures. Two types of substrates were used. In the first substrate (19.Bird E.L. Hakansson K. Pan H. Wigley D.B. Nucleic Acids Res. 1997; 25: 2620-2626Crossref PubMed Scopus (52) Google Scholar), a 82-mer oligonucleotide, 5′-end-labeled using T4 polynucleotide kinase (Biolabs) was annealed to M13mp18 single-strand circles through its central part (nucleotides 24–59) leaving both a 5′ tail (residues 1–23) and a 3′ tail (residues 60–82) unhybridized. Other oligonucleotides, untailed or simply tailed, were also used. In the second type of substrate, described by Tanguy Le Gac et al.(20.Tanguy Le Gac N. Villani G. Boehmer P.E. J. Biol. Chem. 1998; 273: 13801-13807Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), a 5′-labeled 59-mer oligonucleotide was partially hybridized (over 34 nucleotides) to a 90-mer, forming an asymmetric Y-shaped substrate, with a 5′-labeled 25 nucleotide tail and a 3′-unlabeled 56 nucleotide tail. The reaction mixtures (10 μl) contained 20 mm Tris, pH 7.5, 50 mm NaCl, 3 mm MgCl2, 1 mm DTT, 10% glycerol, 1 mm ATP, 0.02 pmol of substrate, and various amounts of the recombinant proteins to test (up to 500 ng). For the second type of substrates, a 27-mer oligonucleotide, complementary to the 90-mer, was added in 4-fold excess to trap the displaced 90-mer. After 10–30 min at various temperatures, the products were analyzed by 12% polyacrylamide non-denaturing gel electrophoresis. PcrA helicase from Bacillus stearothermophilus used as a control was a generous gift of Dr. D. B. Wigley (Oxford). The same assay was used to monitor the relaxation of negatively supercoiled DNA and positive supercoiling, both activities corresponding to an increase of the DNA linking number. The recombinant proteins (0.5–10 ng) were incubated in a 20-μl reaction mixture containing 50 mm Tris, pH 8.0, 10 mm MgCl2, 120 mm NaCl, 0.5 mm DTT, 30 μg/ml bovine serum albumin with 400 ng of negatively supercoiled pTZ18 (Amersham Pharmacia Biotech) at 75 °C for 30 min in the presence or absence of 1 mm ATP. Reactions were stopped by quick cooling on ice and addition of 0.5% SDS, 10 mm EDTA, 5% glycerol, and 0.02% bromphenol blue. The incubation products were analyzed by bidimensional 1.2% agarose gel electrophoresis as described previously (21.Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (88) Google Scholar), with addition of 3 μg/ml chloroquine in the gel and the buffer for the second electrophoresis. For some experiments, monodimensional, 1.2% agarose gel electrophoresis was performed. This assay measures the change in the DNA linking number after stoichiometric binding of a form II DNA with a protein and closure by ligase. In our case, the assay was performed at 75 °C, essentially as described previously (10.Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (39) Google Scholar). Briefly, reaction mixtures (20 μl each in siliconized tubes) contained 20 mm Tris-HCl, pH 7.5, 30 mm NaCl, 25 mm potassium acetate, 10 mm magnesium acetate, 10 mm DTT, 1 mm NAD, 30 μg/ml bovine serum albumin, 33 ng of pTZ 18 form II DNA with an average of one single-strand break per circle, and various amounts of proteins to be tested. Two microliters of a dilution of Taq DNA ligase (Biolabs, 2 units) in 10 mm Tris-HCl, pH 7.5, 100 mm KCl, 20% glycerol were introduced, prior to incubation at 75 °C, as a drop on the inner wall of each siliconized tube but not mixed with the reaction medium. After 10 min of incubation at 75 °C, each tube was quickly agitated to mix the ligase drop with the other components and further incubated for 5 min at 75 °C. This procedure allowed us to avoid variations in the linking number due to temperature changes. After incubation, the tubes were cooled, centrifuged for 30 s, and treated with 50 mm EDTA, 1% SDS, and 0.5 mg/ml proteinase K for 30 min at 50 °C. The reaction products were separated by bidimensional gel electrophoresis, transferred to Hybond N+ membrane (Amersham Pharmacia Biotech), and hybridized with a 33P-labeled pTZ18 probe (random priming, Roche Molecular Biochemicals). The products were revealed either by autoradiography or by using a PhosphorImager (Molecular Dynamics). The entire 1247 amino acid coding sequence of reverse gyrase from S. acidocaldarius was expressed in E. coli as a fusion protein with GST and partly purified by affinity chromatography on glutathione-agarose (see "Experimental Procedures"). In all conditions tested, the level of expression was low, and after the fusion polypeptide was cleaved by thrombin, an additional purification step on phenyl-Sepharose was needed to remove a large part of the remaining contaminants (Fig.1 A). These contaminants, eluted at 25% ethylene glycol (lane 1), are fragments of reverse gyrase since they react with antibodies directed against reverse gyrase in Western blots but have no detectable activity on supercoiled DNA. The main polypeptide, eluted at 60% ethylene glycol, had the expected size for full-length reverse gyrase on polyacrylamide gel electrophoresis (Fig. 1 A, lanes 3 and 4). This recombinant protein was able to fully convert negatively supercoiled plasmid DNA to positive supercoils at 75 °C in the presence of ATP, as shown on the bidimensional analysis of Fig.1 B. This result suggests that, although expressed inE. coli at 37 °C and inactive at this temperature, recombinant reverse gyrase is correctly folded to be active at high temperature as is the enzyme from Sulfolobus. As expected, the mutant enzyme obtained by converting tyrosine 964 into a phenylalanine totally lacks topoisomerase activity, confirming the identity of the active site tyrosine (see Fig. 1 C). Since sequence analysis (12.Confalonieri F. Elie C. Nadal M. de La Tour C.B. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar) suggested that the N-terminal part of reverse gyrase had helicase signatures, and the C-terminal part exhibited topoisomerase I motifs, we designed two polypeptides, covering each half of the whole reverse gyrase sequence. The rationale for the design of these proteins is based on secondary structure predictions and the presence of a highly conserved region in the N terminus of the topoisomerase part (see "Experimental Procedures"). We then expressed both putative domains separately, using the same GST fusion expression system. The helicase-like domain appears as a main band of the expected size, whereas the topoisomerase domain invariably appears as a triplet (Fig.2 A), reminiscent of reverse gyrase degradation fragments (see Fig. 1 A, lane 1). As expected, the putative topoisomerase I domain was able to partly relax a negatively supercoiled plasmid in an ATP-independent reaction at 75 °C (Fig. 2 B, middle). This result, and the complete lack of activity of the putative active site mutant Y352F (data not shown), confirmed that the C-terminal part of reverse gyrase was indeed a topoisomerase domain, as suspected from its sequence. However, this domain exhibited a poor relaxation efficiency (see Fig.2 B, middle, C, panel a, and D, panel g). The putative helicase domain was tested for helicase activity against a variety of substrates formed by untailed, 5′-tailed, 3′-tailed, or doubly tailed oligonucleotides annealed to M13 single-strand circles (see "Experimental Procedures"). As shown in Fig.3, neither the helicase-like domain nor the full-length reverse gyrase exhibited helicase activity on the doubly tailed substrate, whereas the thermophilic helicase PcrA fromB. stearothermophilus (22.Bird E.L. Branningan J.A. Subramanya H.S. Wigley D.B. Nucleic Acids Res. 1998; 26: 2686-2693Crossref PubMed Scopus (91) Google Scholar) was able to partially release the 82-mer oligonucleotide (panel 3). The same result was obtained with the other substrates in a variety of experimental conditions, at 37, 50, 60, or 70 °C (not shown). In another set of experiments, a Y-shaped substrate formed by partial annealing of a 90-mer oligonucleotide to a 5′-labeled 59-mer was used in place of M13 substrates (see "Experimental Procedures") to avoid possible trapping of the proteins by the large amount of M13 single-strand present. Again, in our hands, only the PcrA helicase was able to displace the 59-mer oligonucleotide. We next tried to reconstitute reverse gyrase by simply mixing the two domains at a molar ratio of 1:1. Indeed, incubation of this mixture with plasmid DNA at 75 °C produced ATP-dependent positive supercoiling as efficiently as the full-length enzyme (compare Fig. 2 B, bottom with Fig.1 B, left). In this test, the helicase-like domain alone had no apparent activity on the DNA substrate, whereas the topoisomerase domain exhibited a low relaxation activity (Fig. 2 B,top and middle panels). Remarkably, this latter activity is stimulated by the presence of the helicase-like domain in the absence of ATP (Fig. 2 B, compare middle tobottom). The appearance of a reverse gyrase activity was monitored by adding increasing amounts of the helicase-like domain to a fixed amount of the topoisomerase domain (Fig. 2 C, panels a–f); a plateau of activity was obtained for a 1/1 Hel/Top molar ratio (panel e). Addition of excess "helicase" had no detectable effect (panel f). The same result was obtained by testing increasing amounts of the topoisomerase domain at fixed helicase concentration (not shown). Interestingly, excess of the topoisomerase domain did not change the topoisomer distribution, confirming that this domain was not able to relax positive supercoils. Unexpectedly, when amounts of the topoisomerase domain, low enough to present no visible activity on the substrate (Fig. 2 D,panel g), were supplemented by the helicase-like domain, an efficient reverse gyrase activity was nevertheless reconstituted (panel h). This indicates a strong activation of the topoisomerase activity through interactions between the domains. Finally, reconstitution did not need a preincubation of the mixed domains at low or high temperature, suggesting that when expressed inE. coli, the domains already had a conformation allowing mutual recognition or can acquire it very rapidly when the temperature is increased. Together, these results suggest that the separately expressed domains form a heterodimer that mimics reverse gyrase. The stability of the reconstituted activity toward ionic strength and temperature was compared with that of reverse gyrase. As shown on Fig. 4, the activity of the full-length recombinant enzyme is poor at 30 mmsalt, maximal around 120 mm, and still important at 200 mm, which is similar to the behavior of the endogenous reverse gyrase from Sulfolobus (21.Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (88) Google Scholar). By contrast, the activity exhibited by the mixture of the two domains is highest at low salt and practically nonexistent at 200 mm. The same type of result was obtained when increasing the incubation temperature up to 90 °C; the mixed domains lost their ability to produce positive supercoiling, whereas reverse gyrase retained some activity (not shown). These results suggest a relatively tight association between the domains, which is, however, not strong enough to retain activity in high salt or temperature conditions in the absence of a covalent link between them. The next question that we addressed was how specific the interaction is between the two domains. In other words, supposing that reverse gyrase is a modular enzyme, is it possible to replace either module by a functional equivalent? To test this possibility, we replaced the topoisomerase domain by the type I topoisomerase from Thermotoga maritima, a hyperthermophilic bacterium (23.Huber R. Langworthy T.A. König H. Thomm M. Woese C.R. Sleytr U.B. Stetter K.O. Arch. Microbiol. 1986; 144: 324-333Crossref Scopus (615) Google Scholar, 24.Bouthier de La Tour C. Kaltoum H. Portemer C. Confalonieri F. Huber R. Duguet M. Biochim. Biophys. Acta. 1995; 1264: 279-283Crossref PubMed Scopus (22) Google Scholar). This choice had two advantages as follows: (i) this topoisomerase works at 75–80 °C, and (ii) it has approximately the same size (633 amino acids) as the topoisomerase domain of reverse gyrase (635 amino acids). Incubation of the Thermotogatopoisomerase I with the helicase-like domain of reverse gyrase in the usual reaction mixture did not reveal any positive supercoiling activity (Fig. 5, top), and all conditions tested failed to detect this activity. The same result was obtained when the full-length reverse gyrase mutated in its active site tyrosine (Y964F) was incubated with the Thermotogatopoisomerase I (Fig. 5, bottom). However, in both cases, the presence of the helicase domain, free or included in the reverse gyrase mutant, appeared to stimulate the relaxation activity of theThermotoga topoisomerase, independently of the presence of ATP (compare panels 1 and 2 to 3 and4 and panelss 5 and 6 to 7and 8). The possibility to replace the helicase-like domain by a "true" thermophilic helicase, the PcrA helicase from B. stearothermophilus (22.Bird E.L. Branningan J.A. Subramanya H.S. Wigley D.B. Nucleic Acids Res. 1998; 26: 2686-2693Crossref PubMed Scopus (91) Google Scholar) in the reconstitution assay was also tested and failed (not shown). In a previous work, we showed that in the absence of ATP and at high temperature, the stoichiometric binding ofSulfolobus reverse gyrase to an open circular DNA resulted in a decrease of the DNA linking number after closure by a thermophilic ligase. This was interpreted as an unwinding of the DNA upon reverse gyrase binding (10.Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (39) Google Scholar), a property that is shared by some helicases. We decided first to test this property with the recombinant reverse gyrase mutated on the active site tyrosine (Y964F), so that there was no possible DNA cleavage or interference with the positive supercoiling activity. In addition, it was possible to check the effect of ATP and non-hydrolyzable analogs on this unwinding activity. Incubation at 75 °C of an open circular plasmid with increasing amounts of this mutant in the absence of ATP, followed by covalent closure with the ligase from Thermus, resulted in the progressive appearance of
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