Studies of a Positive Supercoiling Machine

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, 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. dithiothreitol adenylylimidodiphosphate protein-to-DNA molar ratio full-length reverse gyrase a reverse gyrase mutant lacking residues 360–418 a reverse gyrase mutant lacking residues 370–381 a reverse gyrase mutant lacking residues 857–866 polydeoxythymidine Topoisomerases participate in practically every DNA transaction, including transcription, replication, recombination, and chromosomal segregation (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2211) Google Scholar). They change the topological state of DNA by altering its linking number, i.e. the number of times that one strand of the double helix crosses the other. They use a three-step mechanism of cleavage, strand passage, and religation. In the first step, a nucleophilic tyrosine attacks the phosphodiester backbone, cleaving the DNA and leading to a covalent intermediate of protein-DNA termed the “cleavage complex.” Type I topoisomerases cleave one strand of the duplex; type II enzymes cleave both strands. The enzyme, covalently attached to the cut DNA, separates the free ends of the cleaved strand(s) and allows the other strand of the duplex (type I), or another region of duplex (type II), to pass through this gap. The protein then reseals the backbone of the cleaved DNA and releases the product. Although all topoisomerases can relax supercoiled DNA, only prokaryotic gyrase is able to introduce negative supercoils in DNA. This type II topoisomerase uses the energy of ATP hydrolysis to reduce the linking number, leading to an underwound, or negatively supercoiled, product. Until recently, gyrase was the only topoisomerase known to supercoil DNA. Later, another supercoiling enzyme was discovered and named reverse gyrase, because it increases the linking number, leading to overwound, or positively supercoiled, product (2Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar, 3Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (89) Google Scholar). Reverse gyrase is the only topoisomerase known to overwind DNA. So far it has been found exclusively in hyperthermophiles, organisms that live above ∼80 °C (4Bouthier de La Tour C. Portemer C. Nadal M. Stetter K.O. Forterre P. Duguet M. J. Bacteriol. 1990; 172: 6803-6808Crossref PubMed Google Scholar, 5Bouthier de La Tour C. Portemer C. Huber R. Forterre P. Duguet M. J. Bacteriol. 1991; 173: 3921-3923Crossref PubMed Google Scholar). The role of the protein in vivo remains unclear (6Déclais A.-C. Bouthier de La Tour C. Duguet M. Methods Enzymol. 2001; 334: 146-162Crossref PubMed Scopus (10) Google Scholar); one suggestion is that it rewinds the DNA strands in regions of the chromosome that have opened up to allow transcription or replication (7Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google Scholar). Such strand separation at the growth temperatures of hyperthermophiles could be lethal if the strands do not re-associate efficiently. Reverse gyrase belongs to the family of type IA topoisomerases (3Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (89) Google Scholar,8Confalonieri F. Elie C. Nadal M. Bouthier de La Tour C. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar, 9Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (40) Google Scholar, 10Nakasu S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Crossref PubMed Google Scholar), which includes the well-studied topoisomerases I and III fromEscherichia coli. Reverse gyrase differs from other type I enzymes in three key respects. First, whereas type I enzymes can only relax supercoiled DNA, reverse gyrase positively supercoils. Second, reverse gyrase requires nucleotide hydrolysis for this activity (2Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar,10Nakasu S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Crossref PubMed Google Scholar). This property makes reverse gyrase akin to type II topoisomerases in general, all of which require ATP hydrolysis, and specifically to gyrase, which couples ATP hydrolysis to supercoiling. Lastly, in the presence of ATP, reverse gyrase performs the strand passage step unidirectionally: It can increase the linking number of DNA but not decrease it (3Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (89) Google Scholar). In contrast, topoisomerase I can relax either negatively or positively supercoiled DNA (11Kirkegaard K. Wang J.C. J. Mol. Biol. 1985; 185: 625-637Crossref PubMed Scopus (130) Google Scholar), corresponding to an increase or a decrease in linking number, respectively. The N-terminal domain of reverse gyrase provides the ATP energy to drive positive supercoiling by the topoisomerase domain. Understanding how the two domains interact is therefore critical for understanding the enzyme's mechanism. The crystal structure of Archaeoglobus fulgidus reverse gyrase revealed a bimodular enzyme comprising an N-terminal domain with a pair of RecA-like folds homologous to the ATPase domains of helicases, and a C-terminal domain highly homologous to E. coli topoisomerase I (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). The structure immediately suggested a basis for the interaction between the two domains. Residues 352–427 of the N-terminal domain form a “latch” that clamps down over the topoisomerase module. We predicted that the latch would mediate part or all of the communication between the domains. This report explores the role of the latch in the reverse gyrase mechanism. Although the N-terminal domain allows positive topoisomerization in the presence of ATP, it uses the latch to repress topoisomerization in the absence of nucleotide. This study also examines aspects of the protein's DNA-binding behavior, its selectivity for nucleotide cofactor, and the relationship between nucleotide hydrolysis and unidirectional strand passage. An A. fulgidus reverse gyrase expression clone (pRGY1N) was constructed as described previously (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). The clone contains two PCR-induced mutations, Pro-719 → Leu and Leu-1046 → Met. Expression clones containing the Pro-719 → Leu or by or from an clone The topoisomerase domain was from and and in the The with a by and through the of the a as in mutants of reverse gyrase by a to the immediately and of the region to be The with enzymes and cut in the The mutant residues and mutant residues with a and the mutant residues was with The → mutant of reverse gyrase was by the G. S. 1990; 8: Google Scholar). The mutant product was with and and The mutant was by the → the mutant by DNA in E. coli J. Mol. Biol. PubMed Scopus Google Scholar). then containing with to a of and the was to to an in A and at The at at and for at in a The was in A containing The was in and the protein was with and of the A containing was to the the was was to a of The was to a in The reverse gyrase was in and at the for in up in containing of negatively supercoiled and of the The includes by the of with the topoisomerase domain also and but can be with and at then by the of The to in at The was in one for in with for then in the for The was with with at in a of and The was with for in and with a from with and at °C for to the and over a to containing and regions from a study the DNA-binding of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the duplex region is by of polydeoxythymidine to either the or of this The strand was its and with a of to allow The strand was with a of the strand in The duplex has the its corresponding to of the A. fulgidus reverse gyrase The strand is was with and with the as described for the at in with and of the DNA nucleotide was The for at then at with and either for duplex and or for up with of the protein in containing DNA and of the with of the either the or ATP the was for of ATP hydrolysis by full-length reverse gyrase and of hydrolysis was by at °C for the then by to a of and the all been of in was to the which then at °C for at in a A of the was to a and for of this was to of and in a hydrolysis of or by full-length reverse gyrase, the of hydrolysis was by of the of and corresponding which by at °C for the times then by to a of and the of the was and for at with The to a and nucleotide with The full-length reverse gyrase from A. fulgidus was in E. coli (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). This clone contains two Pro-719 to and Leu-1046 to Met. The protein with or the two in positive supercoiling not but to the a clone to the and expression The and mutant reverse described in this report both In the absence of A. fulgidus reverse gyrase at to and reverse M. Jaxel C. Portemer C. Forterre P. Mirambeau G. Duguet M. PubMed Scopus Google Scholar, R. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in the presence of or the protein remains down to In the presence of DNA, is to the protein in A. fulgidus reverse gyrase shows a for its positive supercoiling to the S. and enzymes S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Crossref PubMed Google Scholar, A. J. Biochem. 173: PubMed Scopus Google Scholar, G. Duguet M. Forterre P. J. Mol. Biol. 1984; PubMed Scopus Google Scholar). that this the for regions in the (7Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google Scholar, 9Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (40) Google Scholar, A. S. J. 1990; PubMed Scopus Google Scholar). the DNA strands of a duplex to the DNA is negatively supercoiled, and this allows reverse gyrase to to the DNA. this for DNA in the binding of A. fulgidus reverse gyrase to of DNA was in shows the binding of reverse gyrase to three protein-DNA two that the with in and a that remains in the It is unclear the in the is a protein-DNA or the is to be a the because it in the shows that reverse gyrase also to a duplex examines the enzyme's to to DNA containing both and a duplex region by a either the or the is either the protein the is is to however, is and is of protein and DNA. reverse gyrase to it duplex DNA suggests that the enzyme with both duplex and regions of the the duplex region for containing a and duplex regions of and at by reverse gyrase not suggesting that the duplex region by the protein is at that the protein not require for DNA binding with (7Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google Scholar, 9Jaxel C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (40) Google Scholar, A. S. J. 1990; PubMed Scopus Google Scholar), with the that the of the positive supercoiling the for reverse gyrase to to regions of the Reverse gyrase is type I topoisomerases in its for nucleotide hydrolysis, which it couples to the of DNA. In the to a with the corresponding found of the and only could positive supercoiling by S. reverse gyrase (7Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google Scholar). The other the enzyme to relax DNA but not supercoil This with the that was the only by the hydrolysis of or was (7Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google Scholar). In contrast, the positive supercoiling activity of the A. fulgidus enzyme can be by all positive supercoiling at a for as for S. reverse gyrase (3Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (89) Google Scholar), with for the other The observed for the S. enzyme (7Shibata T. Nakasu S. Yasui K. Kikuchi A. J. Biol. Chem. 1987; 262: 10419-10421Abstract Full Text PDF PubMed Google could be by the in of a of positive whereas the other only not The selectivity of reverse gyrase for was in hydrolysis in the presence of DNA The enzyme ATP and with a number of to a activity of whereas it and at a only above The crystal structure of the enzyme with the nonhydrolyzable ATP adenylylimidodiphosphate suggests that the selectivity for from an interaction between the of the with the of the (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). The between the topoisomerase domain of reverse gyrase and topoisomerase I suggests that reverse gyrase, its E. coli as a DNA enzyme in the absence of nucleotide. topoisomerization by reverse gyrase is observed ATP at molar of protein to DNA The topoisomerase domain its relax DNA in the absence of ATP as for the topoisomerase domain of S. gyrase A.-C. J. F. Bouthier de La Tour C. Duguet M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that the N-terminal domain represses the activity of the topoisomerase domain in the absence of nucleotide. Nucleotide binding is sufficient to topoisomerase activity, because reverse gyrase DNA at in the presence of the N-terminal domain repress topoisomerase The reverse gyrase crystal structure revealed that of the N-terminal a latch over the topoisomerase the to be required for strand passage and topoisomerization A. Mol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). We proposed that binding of both nucleotide and DNA the latch to from the topoisomerase domain (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). this reverse gyrase mutants Mutants constructed lacking the latch residues or the part with the topoisomerase in the crystal structure residues The supercoiling activity of the mutant is in The enzyme, as predicted by shows activity in the absence of nucleotide. the latch is critical for the of topoisomerase activity by the N-terminal domain. and DNA this is in this The mutant to suggesting that topoisomerase is in the region of residues mutants positive supercoiling activity in the presence of ATP, indicating that the latch is not required for coupling nucleotide hydrolysis to positive supercoiling. The latch is also for because both mutants to the full-length protein in in not activity of reverse gyrase mutant lacking residues 370–381 of the up as described in the to In the is as to the of the corresponding of residues 370–381 allows of the topoisomerase activity suggests that the between the latch and topoisomerase domain in the crystal structure is The of this by residues 370–381 of the latch and residues 857–866 of the other of the a mutant lacking residues 857–866 was and for supercoiling activity the full-length enzyme, the mutant not show activity in the absence of nucleotide. This suggests that the latch not interact with residues 857–866 to repress the topoisomerase module. the and mutants in in the supercoiling in the absence of nucleotide hydrolysis, or DNA It is that the form II and III but this for two The to a only with the mutant all the in this study from the E. coli in a and to a of the form II and III require the of a covalent intermediate of reverse gyrase and DNA, because not observed with a mutant a in of the may therefore of that not but topoisomerases the step that DNA hydrolysis by the is not a but the reverse gyrase mutants may be in the step, hydrolysis to to this of the latch allows that the latch nucleotide binding to the topoisomerase domain. it also nucleotide It is not required for because the mutants still positively supercoil DNA in the presence of ATP, but it may still a this ATP hydrolysis activity of the full-length and mutant was in the presence of DNA. This DNA as an to ATP hydrolysis but not as a supercoiling a between the in the of hydrolysis in of supercoiling. to the full-length enzyme, the mutant shows an in ATPase of and the and mutants an of in the region of the latch the of DNA-dependent ATPase activity, the that hydrolysis the state of the In the ATPase of the mutants may that interaction of the latch with the topoisomerase domain is required for hydrolysis to supercoiling. The with the mutant may that this region of the enzyme interact with the latch in a from the interaction required for topoisomerase It is that above a of in the presence of reverse gyrase from to negatively supercoiled the of This to a in the of strand passage from increasing linking number to linking Such a is observed in the presence of ATP the of This may in the absence of hydrolysis, the of the DNA can the in which reverse gyrase performs strand passage. This is an to because it the of reverse gyrase always performs strand passage toward increasing linking number in the presence of ATP (3Forterre P. Mirambeau G. Jaxel C. Nadal M. Duguet M. EMBO J. 1985; 4: 2123-2128Crossref PubMed Scopus (89) Google Scholar). the crystal structure proposed a of strand passage (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar), that the from the that reverse gyrase the DNA strands in a to allow strand passage in only one A been proposed A. S. J. 1990; PubMed Scopus Google Scholar). A for the is by the (6Déclais A.-C. Bouthier de La Tour C. Duguet M. Methods Enzymol. 2001; 334: 146-162Crossref PubMed Scopus (10) Google Scholar). Reverse gyrase is known to DNA binding C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (40) Google Scholar, A.-C. J. F. Bouthier de La Tour C. Duguet M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the domain that the enzyme can this DNA from the of the In DNA that is as a as a the of DNA at the protein binding reverse gyrase increases the linking number of the domain the the is to introduce positive supercoils the the domain the enzyme's to its specificity for strand passage in an domain. to this the strand passage is with the domain the the allows for the that the topoisomerase domain strand passage in either So if reverse gyrase to to an domain of would it increase or decrease the linking this the was A of reverse gyrase was in a supercoiling at °C in the presence of to product This was the of the because the not to not The was the of protein from the C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (40) Google Scholar). an of reverse gyrase mutant was to the and the °C The mutant a in of the DNA to not the enzyme's to and DNA A.-C. J. F. Bouthier de La Tour C. Duguet M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The reverse gyrase to the and that the reverse gyrase would to the strand passage toward increasing linking number, the DNA would as it was at the of the first °C or the strand passage in the the DNA would negatively the indicating that the reverse gyrase strand passage toward increasing linking number the of enzyme, and toward linking number The of reverse gyrase to ATP is with the domain but not with the strand passage The two can be if that the enzyme DNA as described by the domain and nucleotide hydrolysis then a of strand passage. The described show that reverse gyrase to and DNA The enzyme both the and of as topoisomerase In topoisomerase I to cleave the DNA K. G. Wang J.C. Biol. 1984; PubMed Scopus Google Scholar). is the is at K. G. Wang J.C. Biol. 1984; PubMed Scopus Google Scholar), to the of Reverse gyrase a topoisomerase domain with the ATPase domain by a number of helicases, reverse gyrase can its all of the and it ATP and DNA can all of the and but is for S. C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). from with all of the and PubMed Scopus Google Scholar). In contrast, the from can all of the but only hydrolysis of ATP and is in the presence of J. J. F. P. A. C. S. M. J. 1993; PubMed Google Scholar). The N-terminal domain not only the topoisomerase the to positively supercoil DNA ATP but also represses its activity in the absence of nucleotide. The latch specifically the region residues mediates this repression. The that reverse gyrase DNA in the presence of the latch remains in the in the provides that latch binding of both DNA and nucleotide, as suggested from the of (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). the reverse gyrase can positively supercoil DNA in the presence of ATP, but it shows a of DNA-dependent ATP hydrolysis the latch is the latch may be involved but not required coupling nucleotide hydrolysis to the coupling is by other regions of the The crystal structure revealed the of between in the N-terminal domain and in the topoisomerase domain (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). two regions covalently by a between and and in a three of the strands from the first residues of the and the strand residues of nucleotide binding and hydrolysis to the topoisomerase the which to be for binding the DNA and to it (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). The crystal structure suggested that the interaction between the latch and the topoisomerase domain through residues 370–381 of the and residues 857–866 of the of residues 857–866 not the of the latch to repress topoisomerase The between the latch and topoisomerase domain in the crystal structure therefore be with It may be that the latch with the topoisomerase in and the crystal structure only one of It could be that for topoisomerase the latch and topoisomerase domain interact of residues 857–866 but that for coupling of hydrolysis to the interaction This could the mutant shows an in ATPase to that with the The in the latch between the of the and the enzyme with with the of interaction (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). topoisomerase I (11Kirkegaard K. Wang J.C. J. Mol. Biol. 1985; 185: 625-637Crossref PubMed Scopus (130) Google Scholar), reverse gyrase can strand passage in either Nucleotide hydrolysis, however, reverse gyrase to only toward increasing linking Such a role for hydrolysis was not predicted by DNA domains (6Déclais A.-C. Bouthier de La Tour C. Duguet M. Methods Enzymol. 2001; 334: 146-162Crossref PubMed Scopus (10) Google or strand passage (12Rodrı́guez A. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar, A. S. J. 1990; PubMed Scopus Google Scholar). I that of the two of a in the reverse gyrase mechanism and nucleotide hydrolysis drives the between the in the domain and by the described the enzyme to a region of DNA also with the duplex regions and with the duplex allow the enzyme to the DNA C. Nadal M. Mirambeau G. Forterre P. Takahashi M. Duguet M. EMBO J. 1989; 8: 3135-3139Crossref PubMed Scopus (40) Google Scholar, A.-C. J. F. Bouthier de La Tour C. Duguet M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), it an domain at the binding and an domain (6Déclais A.-C. Bouthier de La Tour C. Duguet M. Methods Enzymol. 2001; 334: 146-162Crossref PubMed Scopus (10) Google Scholar). both nucleotide and DNA the latch releases its the topoisomerase strand passage to Nucleotide hydrolysis the enzyme to strand passage toward increasing linking number, of the of the A critical is at hydrolysis in the mechanism. hydrolysis strand passage, or this require how ATP this then the of mutants in the coupling between hydrolysis and supercoiling. Such reverse gyrase uses nucleotide hydrolysis in a as DNA gyrase, the only other topoisomerase known to supercoil DNA. the is how nucleotide hydrolysis reverse gyrase to this a crystal structure of the enzyme with both DNA and nucleotide be I Stock for reverse for this Duguet and of for and and for with the and I to A. and for critical of the and to a for