Reverse Gyrase Functions as a DNA Renaturase

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. 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. Transactions of genetic information in DNA invariably require unwinding and rewinding of DNA double helix, processes usually resulting in creating topological problems. Nature’s solution to such problems is DNA topoisomerases that can reversibly cleave DNA strands and carry out strand passage through an enzyme-mediated DNA break (reviewed in Refs. 1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Crossref PubMed Scopus (2218) Google Scholar, 2Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1916) Google Scholar, 3Corbett K.D. Berger J.M. Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 95-118Crossref PubMed Scopus (351) Google Scholar). Based on the structure and mechanism of topoisomerases, they are grouped into two types, both of which use an active-site tyrosine to form a transient tyrosyl phosphate bond during the step of reversible DNA cleavage. Type I enzymes generate single strand cleavage, and type II enzymes make coordinated breaks in both DNA strands. Type I topoisomerase is divided into two structurally and mechanistically distinct subfamilies. Type IB enzymes, present in all eukaryotes, certain eukaryotic viruses, and some bacterial species (4Krogh B.O. Shuman S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1853-1858Crossref PubMed Scopus (71) Google Scholar), form a 3′-phosphotyrosyl covalent adduct at the DNA cleavage site. They can readily remove either positive or negative supercoils, capable of serving as an efficient swivel during DNA transactions (5Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Crossref PubMed Scopus (622) Google Scholar). Type IA enzymes are ubiquitous, present in eukaryotic, eubacterial, and archaebacterial species. Their enzymatic activity involves strand passage through a protein-mediated DNA gate formed with a tyrosyl linkage to a 5′ phosphoryl group at the cleavage site (6Dekker N.H. Rybenkov V.V. Duguet M. Crisona N.J. Cozzarelli N.R. Bensimon D. Croquette V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12126-12131Crossref PubMed Scopus (115) Google Scholar). These enzymes prefer DNA substrates with a single-stranded region and usually can only relax negative supercoils in a plasmid DNA. Type IA enzymes may have more divergent roles in DNA metabolism besides that as a swivel. Reverse gyrase is the only topoisomerase that can insert positive supercoils into DNA (7Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar). It has two closely linked domains: a helicase domain and a type IA topoisomerase domain (8Confalonieri F. Elie C. Nadal M. de La Tour C. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar, 9Krah R. Kozyavkin S.A. Slesarev A.I. Gellert M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 106-110Crossref PubMed Scopus (46) Google Scholar, 10Declais A.C. Marsault J. Confalonieri F. de La Tour C.B. Duguet M. J. Biol. Chem. 2000; 275: 19498-19504Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The crystal structure of reverse gyrase from Archaeoglobus fulgidus revealed that the structure of the topoisomerase domain is very similar to those from bacterial type IA topoisomerases, and the helicase domain shares the structure with several DNA helicases except for some residues involved in helicase translocation (11Rodriguez A.C. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). Despite the availability of three-dimensional structures, the biochemical mechanism of reverse gyrase remains to be elucidated. Three mechanisms have been proposed. The first is the selective relaxation of the negative supercoils generated behind the translocating helicase moiety (8Confalonieri F. Elie C. Nadal M. de La Tour C. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar). However, there is no evidence that the helicase domain can translocate along DNA because no strand displacement activity was detected with either intact reverse gyrase or with the recombinant helicase domain (10Declais A.C. Marsault J. Confalonieri F. de La Tour C.B. Duguet M. J. Biol. Chem. 2000; 275: 19498-19504Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The second is that upon the unwinding of the DNA by the helicase domain, the reverse gyrase can separate the circular DNA into two topologically independent domains, one with the unwound base pairs, and the other with positive supercoils induced by the unwinding action in the other region (10Declais A.C. Marsault J. Confalonieri F. de La Tour C.B. Duguet M. J. Biol. Chem. 2000; 275: 19498-19504Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The reverse gyrase can renature the unwound region without altering the positive supercoiling in the other domain, thereby leaving behind positive supercoils. How reverse gyrase can segregate the DNA molecule into two topological domains, and how the enzyme can switch between unwinding and rewinding action are not addressed here. The third is that reverse gyrase is capable of direction-specific strand passage, thus generating positive supercoils (11Rodriguez A.C. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). The biochemical basis for this vectorial strand passage is unknown. Our recent experiments with reverse gyrase have revealed several interesting new findings (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Reverse gyrase is able to carry out different supercoiling reactions depending on the nucleotide cofactor. Besides the hallmark positive supercoiling activity by reverse gyrase with ATP, the enzyme is capable of generating hypernegatively supercoiled DNA in the presence of AMPPNP, 2The abbreviation used is: AMPPNP, 5′-adenylyl-β,γ-imidodiphosphate. and a mostly relaxed, but slightly positively supercoiled DNA in the presence of ADP. These supercoiling activities are inefficient and require an excess amount of enzyme to DNA. The final level of supercoiling depends on the enzyme to DNA ratio. This dependence is not because of a kinetic effect, since the reaction rate is the same with different amounts of enzyme in reaction the negative supercoiling by reverse gyrase with is to relaxation by a topoisomerase I to the that negative supercoils are because of the of reverse to the unwound region in DNA. Based on these we that reverse gyrase has for either or DNA, depending on the is ATP or Reverse gyrase with ATP can to the single-stranded region in the DNA, and upon the of ATP to generate the enzyme have a for DNA. This switch in with the strand passage rewinding of the unwound the on the enzyme to DNA, the of final supercoiling is by the enzyme to DNA ratio. In this we have the reactions that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. They supercoil annealing of complementary single-stranded DNA and positive supercoiling of DNA with a single-stranded bubble. These reverse gyrase activities new into biochemical and gyrase was from an A. fulgidus reverse gyrase the from and Stock (11Rodriguez A.C. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). was from the bacterial provided by and A.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of a topoisomerase I from was (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). topoisomerase I was P. J. PubMed Scopus Google Scholar). the enzymes and I from DNA DNA and through double Their from with topoisomerase I P. J. PubMed Scopus Google Scholar). supercoiled DNA substrates generated by DNA with reverse gyrase at an of at the reverse gyrase reaction (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The bubble a circular DNA molecule containing a single-stranded bubble, was by annealing two single-stranded circles from with and the T.S. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). DNA substrates containing a bubble with a but defined size with a of the for bubble substrate. DNA into vectors such that the single-stranded circles from the annealing to DNA with two one from the and the other from the to form a bubble with the length remove the single-stranded bubble with the we used the reaction to the K. R. J. Mol. Biol. PubMed Scopus (46) Google Scholar). The be a circular DNA with a single-stranded bubble of defined size and sequence. the DNA we two DNA for and single-stranded of a size of and with two the DNA and in DNA with a bubble of or in we of They can be to form of which have of and at the The from annealing A and and into the in the for strand and the from annealing and and into for strand The DNA of the single-stranded and regions are in DNA to the of into the DNA from vectors with and can and form a bubble of on one strand and on the other from and form a bubble of 20 and and in a bubble The the bubble are to the size of the bubble at We used of the and strand and in of reverse gyrase reaction containing and reverse gyrase at for step the topological in the of the enzyme was to cleave the at the single site step to generate a The reaction was out in a containing with DNA and of enzyme The reaction was at for and DNA and through a from The reaction in the of a DNA containing a single site and the single-stranded bubble with defined and length the DNA is the species in the reaction we used to from the of the containing the bubble, and the DNA. The DNA the was with to remove and into Reverse reaction was usually out in a containing of DNA nucleotide cofactor, and a amount of reverse gyrase to an from to The reaction was usually out at and by the of and reverse gyrase reaction by in in a containing and supercoiled DNA, we to the and at a of in positively supercoiled DNA, the and of in We also DNA a to the topological linkage between complementary single-stranded circles. The was in an solution containing and The or was out at a of for at The was first with for The was with a solution of for with to DNA by in solution and in The DNA with an DNA we that the plasmid DNA as a there is an of supercoil by of DNA supercoils. we the supercoil relaxation reaction by reverse gyrase substoichiometric of and in the presence of different nucleotide the of reverse gyrase relax DNA in the presence of but can relax DNA with as a and The relaxation reaction with ATP is of the negative supercoils by the first at the hallmark reaction for reverse gyrase is DNA positive supercoiling in the presence of ATP, we have that this reaction requires more than amounts of enzyme. In with a substoichiometric amount of reverse gyrase supercoil DNA The relaxation reaction with as is only in because of the of reverse gyrase to DNA is thereby negative supercoils. A of the reverse gyrase supercoiling reaction is the from to both the negative and positive supercoiling and to (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In an interesting the supercoil relaxation with a substoichiometric amount of reverse gyrase is not to the the from to not the relaxation activity with either or ATP as and The of relaxation with as a is because of the that DNA unwinding is at an the plasmid DNA is in a with unwound regions (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, M. 1993; 21: PubMed Scopus Google Scholar). relaxation reactions out at and the activities not different from those at not that the relaxation reaction by reverse gyrase is not as to as the supercoiling Reverse gyrase is an type IA with both a I and DNA helicase domain present in the and is capable of supercoiling and DNA. negative supercoil relaxation similar to other type IA topoisomerases, requires an unwound in this by negative supercoiling and reaction However, be interesting to reverse gyrase has positive supercoil relaxation and the are for such an We have positively supercoiled DNA substrates, a plasmid DNA DNA with single-stranded bubble and a plasmid with a insert of The bubble DNA was by annealing two single-stranded circles from and as T.S. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). reverse gyrase positive supercoils into the bubble substrate in the presence of ATP supercoil relaxation reactions out with as the cofactor. positively supercoiled are by reverse gyrase and the is to be unwound in a or supercoiled DNA, the positive supercoiling in this DNA base in the region T.S. J.C. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). The positive supercoils in the bubble substrate can be a all positively supercoiled DNA can be completely by a type IB I These demonstrate that in the of supercoil reverse gyrase is similar to other type IA topoisomerases in that can relax supercoiled DNA, and only relax the positive supercoils provided the substrate contains a of DNA of the hallmark reactions for a type I topoisomerase is the of two complementary single-stranded circles through the transient single strand breaks generated by these a type IB and a type IA can mediate this reaction J.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, K. J.C. PubMed Scopus Google Scholar). We have and strands of circular DNA from the and of two single-stranded circles with reverse gyrase in the presence or of The DNA by either or of the complementary circles in the of DNA species than the single-stranded DNA and because annealing complementary to form circular DNA with in both and and with of single-stranded regions However, the two DNA strands are not linked topologically This is by the of in in which the circles and with single-stranded circles and With reverse DNA species than during the of the that topological linkage was However, this strand was efficient with ATP as the The topological linkage between single-stranded circles can be readily with The strand passage between two circles the and the with a single between two strands is the species on the topological linkage DNA and the with the between the strands. supercoiled DNA we that the of is hypernegatively relaxed, and positively supercoiled DNA not and reverse gyrase is inefficient in two single-stranded circles without the presence of either or the at a rate with ATP reactions and We have also the annealing reaction with a type IA topoisomerase I from With an amount of enzyme in of supercoil relaxation I anneals of the single-stranded circles the presence of a helicase domain in a topoisomerase can the strand annealing the helicase domain in reverse gyrase has no DNA translocation we that in the presence ATP has a for single-stranded This activity in reverse gyrase remove base which may the annealing reaction in It is interesting to that in the presence of ATP, reverse gyrase can carry out two distinct but topologically similar annealing complementary DNA strands and positive both of which in an in the with a DNA a DNA supercoiling of plasmid DNA by reverse gyrase is inefficient and requires an excess amount of enzyme. at an of DNA gyrase can than one positive and the of supercoils reverse gyrase molecule at enzyme to DNA (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It is to efficient positive supercoiling in the DNA strand annealing A and because the enzyme to DNA was in these However, the DNA from single strand annealing experiments a single-stranded bubble because of the presence of than in the in the single-stranded circles. the presence of a single-stranded bubble may be for such an efficient positive supercoiling we DNA substrates from the bubble substrate generated from the strand annealing and plasmid We used these DNA substrates in positive supercoiling reactions with a substoichiometric amount of reverse gyrase with the at an of either or the plasmid DNA substrate no in DNA supercoiling a to and With bubble the DNA was positively supercoiled by with an of with the DNA was efficiently supercoiled a with reverse gyrase supercoiling in the DNA was by in the presence of or With positively supercoiled DNA as a with was not The efficient positive supercoiling of a bubble substrate that reverse gyrase can the by in a single-stranded of the for single-stranded region in the bubble substrate used in the experiments is present because of the and is in the length for a single strand bubble, we designed a novel method to a circular DNA substrate with a length and of the single-stranded The reaction and for the It is based on the same as the of the bubble annealing two complementary circles from was into the DNA that the has two single-stranded one from the to a bubble with and the other from the We have also a of into the DNA thus the to remove the bubble and behind a with only the bubble from the for a We designed of to insert into the vectors which in the of a DNA substrate with a bubble size of or nucleotides. The presence of a single-stranded region in these DNA substrates was also by a with either a without the bubble or a plasmid DNA These bubble DNA substrates used in experiments with reverse We first the and bubble substrate in the positive supercoiling reactions A and With an of no in DNA supercoiling In was supercoiled with a similar rate and as the DNA with a larger bubble with The of DNA supercoiling was by in the presence of can DNA thus the DNA the positively supercoiled DNA more such the of DNA between the and the reaction we there is an of and thus 20 supercoils enzyme DNA This a in reactions on the bubble with between and supercoil enzyme plasmid DNA as substrate. However, there is an of a bubble a circular DNA with a bubble is not an efficient substrate. the size of the bubble in supercoiling we the the rate and of supercoiling are for are positive supercoils a and for an not the of supercoiling not a DNA with similar but without single-stranded we used the plasmid DNA as the substrate for the The DNA with similar as those for bubble substrate. This DNA in supercoiling upon reverse gyrase reaction These suggest that the efficient supercoiling reactions require a bubble size of at 20 nucleotides. the in the DNA a single-stranded region for reverse gyrase to we a of the can the of the single-stranded bubble in the supercoiling A of in a DNA substrate is to be the reverse gyrase at a DNA containing of we that the rate and of DNA supercoiling are similar to the bubble substrate of or bubble by with demonstrated an of into the DNA not a similar to those by Reverse gyrase no activity on the the in and there is a of at 20 or more in the size of the single-stranded bubble for efficient positive supercoiling and the single-stranded region can be from either or from a of sequence. Reverse gyrase is an type IA topoisomerase both in of structure and It has two closely linked domains: a helicase domain at and a type IA topoisomerase domain at (8Confalonieri F. Elie C. Nadal M. de La Tour C. Forterre P. Duguet M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4753-4757Crossref PubMed Scopus (135) Google Scholar, 10Declais A.C. Marsault J. Confalonieri F. de La Tour C.B. Duguet M. J. Biol. Chem. 2000; 275: 19498-19504Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, A.C. Stock D. EMBO J. 2002; 21: 418-426Crossref PubMed Scopus (97) Google Scholar). the helicase domain reverse gyrase the helicase domain DNA strand displacement activity (10Declais A.C. Marsault J. Confalonieri F. de La Tour C.B. Duguet M. J. Biol. Chem. 2000; 275: 19498-19504Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The hallmark reaction for reverse gyrase is the of positive DNA supercoils concomitant with the of ATP (7Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar). With reverse gyrase can relax plasmid DNA, but leaving behind a level of positive supercoils (12Hsieh T.S. Capp C. J. Biol. Chem. 2005; 280: 20467-20475Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). a plasmid DNA DNA supercoiling activities of reverse gyrase are and require an excess amount of enzyme relative to DNA. Based on these we a mechanism on how reverse gyrase mediate these topological in DNA. In this we that the of reverse gyrase for either or DNA is by to ATP or ADP. The switch of enzyme from to DNA ATP thus rewinding of single strands. This rewinding activity is by strand passage the enzyme to the of DNA and to positive supercoils. enzyme the of positive DNA supercoiling requires an excess amount of enzyme relative to DNA, and depends on the of enzyme to DNA. We here of efficient strand passage activities of reverse gyrase with a substoichiometric amount of enzyme. to all type IA topoisomerases, reverse gyrase can readily relax supercoiled DNA. In contrast to the supercoiling reactions we supercoil relaxation is not to the reaction This supercoil relaxation activity is only to the supercoiled DNA for the plasmid DNA substrate. reverse gyrase can relax positively supercoiled DNA, provided there is a single-stranded bubble present in the DNA substrate. similar to other type IA topoisomerases, the supercoil relaxation activity of reverse gyrase requires the presence of an unwound or region in the DNA substrate. We also demonstrated here that reverse gyrase can mediate two distinct reactions strand annealing of complementary single-stranded circles and positive supercoiling of DNA with a single-stranded bubble. both and we that single-stranded circles can be readily and into a in the presence of reverse the of two separate circles is during the of the annealing the enzyme mediate strand passage to for the of This strand passage and rewinding activity are efficient with reverse gyrase in the presence of a nucleotide cofactor, ATP that positive supercoiling also strand both of which in an in DNA The positive supercoiling reaction with a bubble substrate is very capable of a level of supercoiling a substoichiometric amount of enzyme. This is in contrast with plasmid DNA for which only a level of positive supercoiling was with an excess amount of enzyme. the single-stranded region in the bubble substrate is to form base pairs, unwound and to as a site for the rewinding action by reverse Besides a single-stranded bubble, the can also as an efficient substrate for the positive supercoiling This is because of the of the at the of a site for reverse gyrase The DNA supercoiling can and positive supercoiling the rewinding of unwound region T.S. J.C. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). have been to one to the of the DNA with base at a and J. Mol. Biol. PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). not for the used in experiments because some of the used in this are not for the in However, is that the positive supercoiling generated here can the rewinding of the A single-stranded bubble, to form base pairs, can also positive supercoiling J. Mol. Biol. PubMed Scopus Google Scholar). This rewinding action by reverse gyrase can thus or the of the The strand annealing and positive supercoiling reactions here the mechanism we for reverse of strand rewinding by a switch of from to DNA, and by the strand passage These reverse gyrase reactions may have important as the reverse gyrase is the only hyperthermophile-specific P. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). biological are involved in the genome the The reverse gyrase at R. J. 2004; PubMed Scopus Google Scholar). The of DNA strands by reverse as demonstrated in the annealing of complementary single strand circles and positive supercoiling in bubble substrates, may be for in the hyperthermophilic Reverse gyrase may thus as a for unwound or bubble and efficiently the strands to DNA on the biochemical basis of the of reverse gyrase with DNA be important for of function and We are to and for a of the of reverse gyrase and We also for on the with