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Originally published In Press as doi:10.1074/jbc.M502739200 on March 22, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20467-20475, May 27, 2005
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Nucleotide- and Stoichiometry-dependent DNA Supercoiling by Reverse Gyrase*

Tao-shih Hsieh{ddagger} and Christopher Capp

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, March 11, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reverse gyrase is a unique type IA topoisomerase that can introduce positive supercoils into DNA. We have investigated some of the biochemical properties of Archaeoglobus fulgidus reverse gyrase. It can mediate three distinct supercoiling reactions depending on the adenine nucleotide cofactor that is present in the reaction. Besides the ATP-driven positive supercoiling reaction, the enzyme can introduce negative supercoils with a nonhydrolyzable analog, adenylyl imidodiphosphate. In the presence of ADP the plasmid DNA is relaxed almost completely, leaving a very low level of positive supercoiling. Surprisingly, the final supercoiling extent for all three distinct reactions depends on the stoichiometry of enzyme to DNA. This dependence is not due to the difference of reaction rate, suggesting that the amount of enzyme bound to DNA is an important determinant for the final supercoiling state of the reaction product. Reverse gyrase also displays exquisite sensitivity toward temperature. Raising the reaction temperatures from 80 to 85 °C, both of which are within the optimal growth temperature of A. fulgidus, greatly increases enzyme activity for all the supercoiling reactions. For the reaction with AMPPNP, the product is a hypernegatively supercoiled DNA. This dramatic enhancement of the reverse gyrase activity is also correlated with the appearance of DNA in a pre-melting state at 85 °C, likely due to the presence of extensively unwound regions in the plasmid. The possible mechanistic insights from these findings will be presented here.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The intertwining of DNA strands in a double helical structure creates interesting topological problems when genetic information is accessed and transacted. DNA topoisomerases are versatile and ubiquitous enzymes that are specifically used to resolve such problems (for review, see Refs. 1 and 2). These enzymes use a tyrosine residue at the active site to reversibly cleave DNA by forming a transient tyrosyl phosphodiester bond. DNA topoisomerases are classified into two types based on biochemical mechanism and structural analysis. Type I enzymes make one single-stranded break at a time; type II enzymes make a concerted double-stranded break and allow another segment of DNA to pass through this transient break. Type I topoisomerases are further grouped into two subfamilies. Type IA enzymes, including bacterial topo1 I, topo III, and eukaryotic topo III, covalently link to the 5' phosphoryl end at the DNA break and allow the complementary strand to pass through this protein-mediated DNA gate (3). Type IB enzymes, primarily consisting of eukaryotic nuclear topo I, work by covalently joining the 3' phosphoryl end at the nick so that the strand with a 5' hydroxyl end can undergo a controlled rotation around the complementary strand (4). Such a difference in the biochemical mechanism between these two subfamilies of type I enzymes suggests that they have distinct intracellular functions. Type IB can readily remove both positive and negative supercoils, thereby serving as an efficient swivel, whereas type IA enzymes have more divergent functions besides a role as swivel. Type IA enzymes have a preference for the single-stranded region in the DNA substrate and in most cases can only relax negatively supercoiled, double-stranded DNA.

Reverse gyrase was first discovered in the hyperthermophilic archaebacterium, Sulfolobus, and has a unique activity in generating positively supercoiled DNA at the expense of ATP hydrolysis (5). It is a type I DNA topoisomerase and has two closely linked domains, a helicase-like domain and a type IA topoisomerase (6–9). The biological functions of reverse gyrase are not completely understood but presumably are intimately linked to its role in generating positive supercoils in DNA. It has been purified from a number of hyperthermophilic eubacteria and archaebacteria, and genome-wide sequence mining revealed that it is the only hyperthermophile-specific protein (10). It is, therefore, likely that reverse gyrase may have important functions in stabilizing the genome structure at high temperature. Genetic knock-out experiments demonstrated that the reverse gyrase mutant is viable but displays significant growth defects at high temperature (11). Recent biochemical experiments suggest that reverse gyrase has an additional function in protecting the DNA strand breakage promoted by exposing DNA to high temperature (12). Therefore, reverse gyrase may have multiple functions in protecting the genome from a hostile growth environment.

A type II DNA topoisomerase, DNA gyrase, has an ATP-driven activity to introduce negative supercoils (13). The vectorial DNA supercoiling process is brought about by the unique right-handed wrapping of DNA around the enzyme and subsequent strand passage through the wrapped DNA segment (for review, see Refs. 2 and 14). The biochemical mechanism for the vectorial supercoiling of a type IA enzyme, reverse gyrase, is still unclear. Interestingly, the helicase domain lacks the DNA translocation activity and cannot displace a short DNA strand from its annealed strand (7). How the ATP binding and hydrolysis are coordinated and used by reverse gyrase to drive the positive supercoiling remains an exciting area of investigation.

We have examined some of the biochemical properties of a recombinant reverse gyrase from Archaeoglobus fulgidus. It is the only enzyme with high resolution structures of the apoenzyme and the enzyme complexed with a non-hydrolyzable analog AMPPNP (9). It consists of two structurally distinct domains, an N-terminal helicase domain and a C-terminal type IA topoisomerase domain. Biochemical studies of this enzyme have been carried out to investigate the role of the "latch" in the enzyme to regulate the topoisomerase activity and ATP hydrolysis (15, 16). In this report we present data on the distinct DNA supercoiling activities of the Archaeoglobus reverse gyrase in the presence of different adenosine nucleotide cofactors. Besides the hallmark positive supercoiling reaction with ATP, reverse gyrase can introduce negative supercoils with AMP-PNP. With ADP, reverse gyrase can more or less relax the plasmid DNA but leave behind a low level of positive supercoils in the DNA product. For all these reactions the final extent of DNA supercoiling depends on the enzyme to DNA ratio, suggesting that DNA binding to the enzyme is critical for the supercoiling state of the reaction product. We propose a model that the reverse gyrase has a structure-specific affinity for DNA depending on its association with ATP or ADP, and this distinct affinity of reverse gyrase for single versus double-stranded DNA controls the direction of DNA supercoiling in these reactions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA and Enzymes—Plasmid DNA pGFP-C1 (Clontech, Palo Alto, CA) is 4.73 kilobases in length. It was purified through double banding in CsCl/ethidium bromide density gradient ultracentrifugation. After the removal of ethidium by butanol extraction, the DNA sample was dialyzed exhaustively into TE (10 mM Tris-HCl, pH 7.9, 0.1 mM EDTA) and used as a substrate for reverse gyrase reactions. Singly nicked DNA was prepared by digesting DNA with pancreatic DNase I in the presence of saturating amounts of ethidium bromide as described previously (17). Relaxed DNA was prepared by treating the plasmid DNA with recombinant Drosophila topoisomerase I (18). Both nicked and relaxed DNA were purified by phenol extraction and ethanol precipitation after the enzymatic reaction. Reverse gyrase was purified from a bacterial strain expressing cloned A. fulgidus reverse gyrase (9) following the published procedure with minor modifications. The purified protein was further chromatographed through a Porus HQ column (PerSeptive Biosystems, Farmington, MA). The pooled fractions were dialyzed into a buffer containing 15 mM KPi, pH 7.2, 50 mM NaCl, 0.1 mM EDTA, 0.2 mM dithiothreitol, and 50% glycerol and stored at –20 °C. This recombinant Archaeoglobus reverse gyrase contained two mutations introduced during molecular cloning: Pro-719 to Leu and Leu-1046 to Met (16). The crystal structure and most of the published biochemical experiments were from this recombinant enzyme. Although these mutations allow for efficient expression in Escherichia coli cells, they do not appear to alter the biochemical properties of DNA supercoiling when compared with the wild type protein (15, 16). To isolate a thermostable topoisomerase I, we cloned the top1 gene from Thermotoga maritima by obtaining top1 gene through PCR similar to the method described by Viard et al. (19). Thermotoga top1 was cloned into pET23b vector (Novogen, CT) such that a hexahistidine tag was fused in frame to the C terminus. The cloned vector was sequenced and transformed into the T7-expression strain BL21(DE3)LysS. T. maritima topoisomerase I (Tm topo I) was induced and purified through a nickel nitrilotriacetic acid column. Taq DNA ligase was purchased from New England Biolabs (Beverly, MA).

Reverse Gyrase Reaction—A typical reaction mixture for reverse gyrase contains 0.45 µg of DNA and reverse gyrase in a 30 µl solution of 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 50 µg/ml gelatin, and 1 mM adenine nucleotide cofactor when required. A drop of mineral oil was added to cover the surface of the solution, and the reaction was initiated by immersing the reaction tube in a water bath set at either 80 or 85 °C. The reaction was terminated by adding EDTA and SDS to a final concentration of 10 mM and 0.1%, respectively. If the reaction mixture was to be analyzed by agarose gel electrophoresis, sucrose and tracking dyes (bromphenol blue and xylene cyanol) were added to a concentration of 5 and 0.01%, respectively. Because of the difficulty in manipulating the reaction mixture at high temperature, the time-course reaction was carried out in aliquots of separate reaction tubes. A grand reaction mixture complete with all ingredients was constituted and separated into aliquots in separate reaction tubes. After adding the mineral oil, they were immersed in a water bath. At intervals of 3.5, 7, 15, 30, 60, or 120 min they were removed, and reactions were stopped.

DNA Ligase Reactions—DNA ligation of singly nicked DNA was carried out under conditions identical to those for reverse gyrase reactions except for the cofactors. The reaction mixture contained 0.5 µg of DNA and 7.5 units of Taq DNA ligase in 30-µl solutions of 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 50 µg/ml gelatin, and 1 mM NAD. The reaction solution was layered with a drop of mineral oil, and the reaction was carried out by immersing in a water bath with temperature ranging from 50 to 85 °C for 15 min. Analysis of DNA products from reactions with 7.5 or 30 min gave the same amount and pattern of DNA topoisomer distribution as that from the 15 min reaction, indicating that the reaction was complete under our reaction conditions.

Tm Topo I Relaxation Reaction—DNA relaxation reactions were carried out under conditions similar to those for reverse gyrase except for no cofactors were added. One unit of enzyme was defined as the activity capable of relaxing 0.4 µg of plasmid DNA in 30 min at 75 °C.

Agarose Gel Electrophoresis—DNA products were analyzed by electrophoresis in 1% agarose under a voltage gradient of 1.5 V/cm for 16 h in room temperature. The sign of supercoiling for DNA products was determined by the following methods. Gel electrophoresis could be carried out at 4 °C. If DNA topoisomers gained mobility upon lowering the running temperature from ambient room temperature to 4 °C, they were negatively supercoiled, and a reduced mobility would indicate they were positively supercoiled. DNA topoisomers not resolved under either of these conditions were analyzed by electrophoresis in the presence of varying chloroquine concentrations ranging from 30 to 120 µM. Hypernegatively supercoiled DNA, which failed to be completely resolved by electrophoresis in 120 µM chloroquine, were analyzed by running the gel in the presence of 0.2 µg/ml ethidium bromide. We were able to resolve all the DNA products into topoisomer bands under one of the aforementioned conditions. At the end of electrophoresis the gel was first stained in a solution of 0.3 µg/ml ethidium bromide for 30 min, then irradiated with intense UV light to promote nicking of DNA. The gel was stained again for an additional 30 min, after which it was destained for 1 h. For the gels with high concentrations of chloroquine, the destaining process could take up to 3 h. The stained/destained gel was imaged with EpiChem system (UVP Bioimaging System, Garland, CA). The digitized images could be further processed for analysis by LabWorks Imaging and Analysis Software (UVP Bioimaging System).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nucleotide-dependent DNA Supercoiling—Earlier works have shown that positive DNA supercoiling by reverse gyrase requires a nucleoside triphosphate cofactor with a preference or specificity for ATP and dATP (16, 20). To investigate whether nucleotide cofactors other than ATP have any effect on the reactions by reverse gyrase, we examined the reactions carried out with different forms of adenosine nucleotide using a plasmid DNA as the substrate (Fig. 1). In the absence of exogenous cofactor or in the presence of AMP there is a slow relaxation of the plasmid DNA (Fig. 1, lanes 5–10 and 11–15), evidenced by the appearance of topoisomers at the longest time point. As expected, the presence of ATP promotes the positive supercoiling reaction (Fig. 1, lanes 21–25). Interestingly, ADP enhances the relaxation reaction, which is complete within 3.5 min when the first time point was taken (lanes 16–20). The DNA supercoiling reaction in the presence of ATP is also preceded by a rapid relaxation, giving rise to the appearance of a biphasic reaction; that is, a fast relaxation of the plasmid negative supercoils followed by a slower insertion of positive supercoils. Using gel electrophoresis in the presence of various chloroquine concentrations, we determined that the plasmid substrate used here, a DNA with 4.7 kilobases in length, contains about 30 supercoils (superhelical density {sigma} = –0.06). Therefore, there is a rapid relaxation within 3.5 min to remove 30 negative supercoils followed by a slow insertion of 5 positive supercoils in the reaction with ATP. Further analysis of the relaxation reaction rates in the presence of ADP, ATP, or AMP-PNP demonstrates that the relaxation rate is faster than the supercoiling by at least 2 orders of magnitude (data not shown).

In the presence of a nonhydrolyzable analog of ATP, AMP-PNP, one also observes a partial relaxation of DNA at the first time point followed by a slower DNA supercoiling reaction (lanes 26–30). To address whether the supercoiling is positive or negative, we analyzed the reaction products with gel electrophoresis in the presence and absence of 30 µM chloroquine (lower and upper panels of Fig. 2, respectively). As a reference we loaded in the same gels the products from the reactions with no cofactor, AMP, and ATP. In the presence of a DNA unwinding agent, chloroquine, plasmid DNA is slightly negatively supercoiled and resolved as distinct topoisomer species (leftmost lane, SC, Fig. 2). DNA from reactions with no cofactor or with AMP is partially relaxed, running as slightly negatively supercoiled in the early time points then as slightly positively supercoiled in later time points. In contrast, the relaxed DNA from reactions with ATP in early time points and the positively supercoiled product in the later time points ran as highly positively supercoiled species. The products of the AMPPNP reaction have similar mobilities to the DNA from no cofactor or AMP reactions, suggesting that they are negatively supercoiled. It is interesting to note that depending on the presence of cofactors, reverse gyrase can carry out all three distinct topoisomerization reactions, namely relaxation, positive, and negative DNA supercoiling.



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  		FIG. 1.
Reverse gyrase reaction time course at 80 °C. Reaction products were sampled at intervals of 3.5, 7, 15, 30, and 60 min for control reactions without enzyme (lanes 1–5) and for reverse gyrase reactions in the absence of cofactor (lanes 6–10) and in the presence of 1 mM AMP (lanes 11–15), 1 mM ADP (lanes 16–20), 1 mM ATP (lanes 21–25), and 1 mM AMPPNP (lanes 26–30). The marker lanes for either supercoiled plasmid DNA (SC) or relaxed DNA (RC) are at the left end of the gel. The molar ratio of the enzyme to DNA in these reactions was 12.

 
Final Extent of Supercoiling Depends on Enzyme/DNA Stoichiometry—The enzyme/DNA molar ratio for the reactions shown in Fig. 1 was 12, and only limited positive supercoiling was observed in the presence of ATP. To check if a higher amount of enzyme added to the reaction can bring about more positive supercoiling, we carried out the reactions with enzyme/DNA ratios ranging from 47 to 0.84 (Fig. 3). DNA positive supercoiling increases with the enzyme/DNA ratio in the reactions with ATP (lanes 1–5, Fig. 3). The negative supercoiling of reverse gyrase in the presence of AMPPNP also depends on the stoichiometry of the enzyme (lanes 6–10, Fig. 3). These results are unexpected since all the other topoisomerases studied thus far, including DNA gyrase, act catalytically, and the final state of DNA supercoiling is, thus, not sensitive to enzyme stoichiometry. To examine whether dependence of supercoiling on the enzyme/DNA ratio is simply due to a difference in the rate of supercoiling in the presence of varying amounts of reverse gyrase, we carried out the following two experiments. First, time course experiments showed that at both high and low enzyme/DNA ratios, the reactions approached a final supercoiling state after incubation for 1 h (Fig. 4). This is true for reactions with ATP and AMPPNP (Fig. 4, lanes 1–10 and 11–20, respectively). Because the results showed in Fig. 3 were obtained with reactions carried out after a 1-h incubation, the difference in supercoiling state is likely not due to reaction rate. Second, we carried out the reactions under conditions of either constant DNA concentration or constant enzyme concentration and obtained similar results (Fig. 5). In the experiments presented in Figs. 3 and 4, DNA concentration was maintained the same but with varying enzyme concentrations. This type of experiment was repeated again as shown in Fig. 5 (lanes 5–8). In these experiments, DNA concentration was held constant, but enzyme concentrations ranging from 0.08 to 0.7 µM were used to give an enzyme/DNA ratio from 11 to 94. As expected, the final positive supercoiling depends on the stoichiometry. Interestingly, if reactions were carried out at an enzyme concentration of 0.7 µM and varying DNA concentration to give the same range of enzyme/DNA ratio (lanes 1–4, Fig. 5), the final supercoiling states are identical to those obtained using variable enzyme concentrations. Experiments similar to that shown in Fig. 5 were done using AMPPNP as a cofactor, and they demonstrated that the final negative supercoiling state also depends on the enzyme/DNA ratio (data not shown).



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  		FIG. 2.
Analysis of DNA products of reverse gyrase reactions by agarose gel electrophoresis in the absence (upper panel) and presence of 30 µM chloroquine (lower panel). The products from a reaction time course identical to those shown in Fig. 1 were analyzed to determine the sign of the supercoiling state of the DNA. In 30 µM chloroquine (CLQ), the plasmid DNA (SC) was run as a group of partially relaxed topoisomer species (left end of the gel), whereas the partially positively supercoiled DNA from the ATP-reverse gyrase reaction migrated as highly positively supercoiled species with a fast electrophoretic mobility. Notice that the topoisomer species can be observed in the gel with chloroquine for the products from reactions with AMP or AMPPNP, suggesting that they are negatively supercoiled.

 



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  		FIG. 3.
Stoichiometry-dependent DNA supercoiling by reverse gyrase (RevGyr). Reverse gyrase can carry out positive supercoiling in the presence of ATP (lanes 1–5) and negative supercoiling with AMP-PNP (lanes 6–10). The enzyme/DNA ratios in these reactions were 47, 23, 7.6, 2.5, and 0.84 (lanes 1–5 and lanes 6–10). The extent of supercoiling increases as the stoichiometry increases. The starting material was loaded at the left end of the gel (SC).

 



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  		FIG. 4.
Stoichiometry dependence of DNA supercoiling is not due to different reaction rates. Reactions were sampled at intervals of 3.5, 7, 15, 30, and 60 min for reverse gyrase with ATP (lanes 1–10) or with AMPPNP (lanes 11–20). The enzyme/DNA ratio was either 47 (lanes 1–5 and lanes 11–15) or 0.84 (lanes 6–10 and lanes 16–20). At either enzyme/DNA ratio the reaction reached its end point by 60 min. The starting material plasmid DNA (SC) and a marker nicked DNA (NC) were loaded at the left end of gel.

 



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  		FIG. 5.
Stoichiometry dependence of DNA supercoiling is independent of enzyme concentrations. ATP-driven positive supercoiling reactions were carried out with different enzyme/DNA ratios of 94, 46, 23, and 11 at either a constant enzyme concentration of 0.7 µM (lanes 1–4) or constant DNA concentration but different enzyme concentrations of 0.7, 0.35, 0.17, and 0.08 µM (lanes 5–8). Triangles on top of the gel denote the change of enzyme to DNA ratio. Notice that even at an enzyme concentration of 0.7 µM, the extent of supercoiling was identical to a reaction with the same enzyme/DNA ratio but with a nearly 10-fold lower enzyme concentration of 0.08 µM (lane 4 and 8). The starting material plasmid DNA (SC) is in the rightmost lane.

 
In the experiments shown in Fig. 1 the final products of reverse gyrase reactions in the presence of ADP are relaxed DNA. We have tested if the final supercoiling state of DNA products from such reactions also depends on enzyme/DNA stoichiometry (Fig. 6). In a series of reactions similar to those carried out in Fig. 3 using ADP as a cofactor, the final supercoiling state changes as the enzyme/DNA ratio varies from 47 to 1.2 (lanes 1–5, Fig. 6A). However, the range of supercoiling change is much smaller than that obtained with ATP or AMP-PNP. To ascertain whether the supercoiling change upon increasing reverse gyrase concentrations is positive or negative, we analyzed the same set of reaction products by gel electrophoresis in the presence of a low concentration of the intercalator agent, chloroquine (Fig. 6, panel B). We used the DNA sample from reverse gyrase positive supercoiling reaction with ATP (enzyme/DNA ratio of 7.6) as a control (lane 6 of Fig. 6, A and B). There is a shift of about +4 superhelical turns in the topoisomer species analyzed in the presence of chloroquine. This small shift in DNA supercoiling was not sufficient to bring the plasmid DNA into the range of resolved topoisomers as those shown in Fig. 2, but it could resolve the topoisomers from all the samples of ADP/reverse gyrase reactions (lanes 1–5, Fig. 6B). The higher the amount of enzyme, the more positively supercoiled the product is, and thus, the faster the mobilities of the topoisomers in the chloroquine gel (Fig. 6B). Therefore, in the presence of ADP, reverse gyrase can relax negative supercoils in the plasmid DNA and results in positive supercoiling. The level of positive supercoiling is very low but still depends on the enzyme stoichiometry.



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  		FIG. 6.
Stoichiometric dependence of low level positive supercoiling by reverse gyrase with ADP. Reaction products of reverse gyrase with ADP were loaded in the gel without or with 2 µM chloroquine (panels A and B, respectively). The enzyme/DNA ratios for the reactions were 47, 23, 7.6, 2.5, and 1.2 (lanes 1–5). Positively supercoiled DNA from the ATP/reverse gyrase reaction was loaded (lane 6) to calibrate the supercoiling shift under these two conditions of gel electrophoresis. Notice that the limited shift in supercoiling due to a low concentration of chloroquine is insufficient to resolve the plasmid DNA (SC) in the range of relaxed topoisomers.

 
Reverse Gyrase Reactions at 85 °C—The supercoiling reactions for Archaeoglobus reverse gyrase described thus far and in earlier published works (15, 16) were carried out at 80 °C. The optimal growth temperature for A. fulgidus was reported to be between 80 and 85 °C (21, 22). We were interested in analyzing the temperature sensitivity of the supercoiling reactions within the optimal temperature range for this enzyme. To examine the temperature dependence of reverse gyrase reactions in the presence of different cofactors, we performed experiments similar to those shown in Fig. 1, except at 85 °C (Fig. 7). When analyzed by agarose gel electrophoresis without intercalating reagents the products from reactions without cofactor or with AMP appear to have the same mobility as the starting material plasmid DNA (lanes 6–15, Fig. 7). Like the reactions at 80 °C, reactions with ADP result in rapid relaxation (lanes 16–20) and with ATP result in relaxation followed by positive supercoiling (lanes 21–25). However, the reactions with AMPPNP result in products with mobility similar to plasmid DNA (lanes 26–30).

To check the supercoiling state of the DNA products, we analyzed them by gel electrophoresis in the presence of increasing concentrations of chloroquine (Fig. 8, panels A–D). In the presence of 30 µM chloroquine, plasmid DNA was partially relaxed, and the resolved topoisomers ran just ahead of the nicked DNA species (Fig. 8B). The DNA products from early time points of reactions either without cofactor or with AMP or AMPPNP showed similar mobility to plasmid DNA, suggesting that they are negatively supercoiled. In contrast, the DNA products from all time points of the reactions with ATP ran as a tight band ahead of the relaxed species. This is consistent with these DNA products being positively supercoiled.



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  		FIG. 7.
Reverse gyrase reaction time course at 85 °C. Reaction products were sampled at intervals of 3.5, 7, 15, 30, and 60 min for control reactions without enzyme (lanes 1–5) and for reverse gyrase reactions in the absence of cofactor (lanes 6–10) or in the presence of 1 mM AMP (lanes 11–15), 1 mM ADP (lanes 16–20), 1 mM ATP (lanes 21–25), and 1 mM AMPPNP (lanes 26–30). The marker lane for supercoiled plasmid DNA (SC) is at the end of the gel. The mole ratio of the enzyme to DNA in these reactions was 1.0.

 
The DNA products from later time points of all reactions showed much greater electrophoretic mobility (Fig. 8B). It is possible that they are either positively supercoiled, like the products from reactions with ATP, or highly negatively supercoiled, thus requiring a higher concentration of chloroquine to resolve them into relaxed species. To test this, we ran these DNA products in the presence of 60 and 120 µM chloroquine (Fig. 8, C and D, respectively). The later time point products from reactions without cofactor or with AMP can be resolved into topoisomers running close to the relaxed species in the gel with 60 µM chloroquine (Fig. 8C), and they are converted into fully relaxed species with 120 µM chloroquine (Fig. 8D). The later time point products from reactions with AMPPNP only begin to be resolved into topoisomers with 120 µM chloroquine. They can be resolved as relaxed species in the presence of 0.2 µg/ml ethidium (data not shown). These results, therefore, demonstrate that at 85 °C either without cofactor or with AMP, reverse gyrase can convert the DNA into products with slightly more negative supercoils than plasmid DNA. However, reactions with AMPPNP can convert DNA into hypernegatively supercoiled species.

Besides generating hypernegatively supercoiled DNA in the presence of AMPPNP, reverse gyrase reactions at 85 °C also differ from those at 80 °C in another important aspect. The apparent specific activity of reverse gyrase is much higher at 85 °C. Although giving rise to DNA products with a similar extent of supercoiling in the presence of ATP, the enzyme/DNA ratio in reactions shown in Fig. 7 is 1.0, as compared with a ratio of 12 for reactions shown in Fig. 1. Similar to the results obtained at 80 °C, the final extent of DNA supercoiling for reverse gyrase reactions at 85 °C depends on the enzyme to DNA ratio. The extent of both positive supercoiling with ATP and the negative supercoiling with AMPPNP increases as the enzyme/DNA stoichiometry increases (data not shown). With the same enzyme/DNA ratio, the extent of supercoiling from reactions at 85 °C is always much more than from 80 °C.

In contrast to the reactions at 85 °C, reverse gyrase reactions at 75 °C are less proficient than those at 80 °C. The final extent of supercoiling is much lower than that at 80 °C at the identical enzyme/DNA ratio (data not shown). It is interesting to note that a change of reaction temperatures by 5 °C near the optimal growth temperature of A. fulgidus can lead to a dramatic change in both the activity and product supercoiling state of the reverse gyrase reactions. This unique dependence on the temperature may also have important biological functions for this enzyme in the hyperthermophiles.

Extent of DNA Supercoiling Induced by Reverse Gyrase—To determine the number of positive supercoils introduced by reverse gyrase, we need a reference DNA that is fully relaxed at the same temperature and buffer conditions as the reverse gyrase reaction. We prepared DNA with a single nick per molecule and rejoined the nick with Taq DNA ligase in the same buffer conditions as the reverse gyrase reaction, except for the cofactors. The ligation reactions were carried out at a series of temperatures ranging from 50 to 85 °C (panels C–G, Fig. 9). Ligation products at 80 °C clearly follow a Gaussian distribution of DNA topoisomers (panel F, Fig. 9). Analyzing the DNA products by gel electrophoresis under different conditions (e.g. at 4 °C or with low concentrations of chloroquine) also reveals Gaussian distributions for DNA ligated at 50–70 °C (data not shown). Earlier experiments analyzing DNA ligated between 14 and 29 °C (23) and at temperatures up to 79 °C (24) showed that DNA topoisomers are present as a Gaussian distribution. The difference between these Gaussian distributions corresponds to a decrease in the average linking number of the topoisomers as the DNA unwinds with increasing temperatures. Interestingly, ligation products at 85 °C not only give the similar set of topoisomers to those from 80 °C but also a supercoiling density close to that of plasmid DNA (panel G). This may be due to extensive helical unwinding present at the pre-melting state at 85 °C. This structural transition in the pre-melting state may account for the enhanced reverse gyrase activities observed at 85 °C (see next sections). Using a different plasmid DNA substrate and slightly different buffer conditions, negatively supercoiled DNA product was also observed with ligation at 83 °C, which was attributed to pre-melting of DNA under such conditions (24).

We carried out reverse gyrase reaction in the presence of ADP, ATP, and AMPPNP at either 80 °C (Fig. 9, panels H–J) or 85 °C (panels K–M). In comparing topoisomer distributions between ligase products and positively supercoiled DNA from the reverse gyrase reaction at 80 °C (panels F and I), the linking number shift between these reaction products was estimated to be {Delta}Lk = +9. Notice that under conditions of gel electrophoresis, reverse gyrase products are positively supercoiled whereas ligase products are negatively supercoiled (solid versus dotted line in panel I). The enzyme/DNA ratio in this experiment is 10, suggesting that on average a 0.9 positive turn is introduced by each reverse gyrase in the presence of ATP. However, data shown in Fig. 4 (lanes 1–5) indicate that the average linking number shift per reverse gyrase is only about {Delta}Lk = +0.1 per enzyme when the enzyme/DNA ratio changes from 23 to 47. Although the biochemical basis of this difference is unclear, it is possible that positive DNA supercoiling by reverse gyrase is initiated through the binding of the enzyme to a limited region on the plasmid DNA where base pairs are already unwound. These unwound regions presumably correspond to the AT-rich sequences surrounded by regions with a higher GC content. Additional enzymes can only introduce limited change in DNA structure extended from these unwound regions, thus bringing about much less change in supercoiling. The initial positive supercoiling per enzyme is, thus, higher than that at later stages with additional enzyme. This is consistent with the observation of enhanced reverse gyrase activities at 85 °C (panel L). The enzyme/DNA ratio for the 85 °C reaction is 1, only of the enzyme used in 80 °C. But the average linking number of DNA products is more positive than that of the 80 °C reaction by about 3 (compare panel L with panel I). Therefore, it is possible that reverse gyrase binding to the more underwound structure of DNA at 85 °C can lead to a larger change in DNA supercoiling.



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  		FIG. 8.
Analysis of DNA supercoiling by gel electrophoresis with different concentrations of chloroquine. Identical sets of reaction products were loaded onto gels without chloroquine (CLQ; panel A) or with 30 µM (panel B), 60 µM (panel C), and 120 µM (panel D) of chloroquine. The sets of samples from time course experiments were identical to those shown in Fig. 7. Notice that DNA products are more negatively supercoiled than the plasmid DNA control (SC, leftmost lane in each gel) for the reactions without cofactor or with AMP or AMPPNP at later time points of 30 and 60 min. At the highest concentration of chloroquine, 120 µM, the samples from reactions with AMPPNP, which were hypernegatively supercoiled, started to show partially relaxed topoisomers.

 
In the presence of AMPPNP, reverse gyrase can introduce negative supercoils into DNA at 80 and 85 °C (panels J and M, respectively, and also data shown in Figs. 2 and 8). The overlapped tracings of DNA topoisomers from ligase and reverse gyrase reactions also suggest that they are negatively supercoiled (dotted and solid line in panel J). At 80 °C with an enzyme/DNA ratio of 10, the linking number change is about {Delta}Lk = +10, corresponding to at least 1 negative supercoil per reverse gyrase. Interestingly, the negative supercoiling activity by reverse gyrase is also greatly stimulated at 85 °C, and DNA products from an enzyme/DNA ratio of about 1 are hypernegatively supercoiled (panel M; see also Fig. 8). Again, the partially unpaired DNA structure in the pre-melting state might be responsible for generating this.

The experiments presented here also demonstrate that reverse gyrase can readily relax negatively supercoiled DNA in the presence of ADP. However, a close comparison of the reverse gyrase DNA products with the ligated DNA showed that reverse gyrase can introduce a limited amount of positive DNA supercoils into DNA (panels H and K). Although the DNA ligation products are negatively supercoiled under gel electrophoretic conditions as in Fig. 9, the staggered densitometric tracings of the reverse gyrase products in the presence of ADP indicate that they are slightly positively supercoiled (dotted and filled lines in panels H and K). This is also consistent with gel electrophoretic analysis at low concentrations of chloroquine, as shown in Fig. 6. However, the number of supercoils introduced by reverse gyrase with ADP is much less than with ATP. At 80 °C and an enzyme/DNA ratio of 10, about 4 positive supercoils are introduced, corresponding to about 0.4 supercoils per enzyme (panel K). Similar to the reactions with ATP and AMPPNP, reactions with ADP are also stimulated at 85 °C. One can observe DNA products with slightly more positive supercoils at a much lower enzyme/DNA ratio (enzyme/DNA ratio of 1 at 85 °C versus that of 10 at 80 °C, panels K and H, respectively).

Negative Supercoiling Generated by Reverse Gyrase in the Presence of AMPPNP Is Constrained—The stoichiometry dependence of reverse gyrase supercoiling suggests that protein binding plays a critical role in the supercoiling process. If the DNA supercoiling is produced as a result of conformation-specific binding by a protein, it will be constrained and cannot be removed by the relaxing activity from a topoisomerase. The negative supercoiling by reverse gyrase/AMPPNP provides an opportunity to test this hypothesis. Such experiments would require an efficient and thermostable topo I. We use topo I from T. maritima because its activity is optimal at 75 °C and remains robust over a wide temperature range (19), to test if it can remove the negative supercoiling generated by reverse gyrase/AMPPNP (Fig. 10). At 80 °C, the stoichiometry-dependent negative supercoiling (lanes 6 and 7) is resistant to the relaxation by the addition of an excess amount of Tm topo I (lanes 8 and 9). At 55 °C the same amount of reverse gyrase has no activity in changing the plasmid DNA supercoiling (lanes 2 and 3), and Tm topo I can relax the DNA supercoils (lanes 4 and 5). The negative supercoils produced by reverse gyrase at 80 °C are also retained after the reaction mixtures were shifted to 55 °C (lanes 10 and 11) since reverse gyrase is inactive at this temperature. But Tm topo I can readily relax these negative supercoils under such conditions (lanes 12 and 13). These data suggest that reverse gyrase does not inactivate Tm topo I, and the retention of DNA supercoils is presumably due to the constraint of supercoiling by reverse gyrase. Works presented here and in an earlier paper (25) suggest that conformation-specific binding by reverse gyrase is very sensitive to temperature. At 55 °C, the loss of conformation specific binding by reverse gyrase results in unconstrained DNA supercoiling and its relaxation by Tm topo I.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The experiments presented here reveal a number of unique features in the reactions of reverse gyrase and may shed light on its mechanism. Reverse gyrase can change the state of DNA supercoiling depending on adenine nucleotide cofactor. Besides the hallmark reaction of positive supercoiling with ATP, the enzyme can insert negative supercoils into plasmid DNA in the presence of AMPPNP. With ADP as a cofactor, reverse gyrase can also relax the plasmid DNA but leave behind a low level of positive supercoiling. These reactions are exquisitely sensitive to temperature, greatly stimulated by an increase of 5 °C within the range of optimal growth temperature for the hyperthermophilic archaebacterium, Archaeoglobus. Furthermore, the final level of DNA supercoiling for all the reverse gyrase reactions depends on the enzyme/DNA ratio, suggesting that enzyme/DNA binding is a determinant for the final supercoiling state. This notion is also supported by the data that negative supercoiling by reverse gyrase and AMPPNP is constrained and cannot be removed by the addition of another topoisomerase I.



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  		FIG. 9.
Densitometric tracings of ligase and reverse gyrase reaction products analyzed by agarose gel electrophoresis. Samples of ligation products by Taq ligase reactions at 50–85 °C are shown in panels C–G. Panels H–J and K–M show the products from reverse gyrase reactions at 80 and 85 °C, respectively. The cofactors present in the reverse gyrase reactions were ADP (panels H and K), ATP (panels I and L), and AMPPNP (panels J and M). Panels A and B are plasmid supercoiled and nicked DNA, respectively. The dotted lines present in the tracings for reverse gyrase products (panels H–M) are the reference DNA samples from ligase reactions at either 80 or 85 °C. SC, supercoiled DNA.

 
There are at least three biochemical mechanisms proposed for reverse gyrase. The first is a coupled helicase translocation and selective relaxation of negative supercoiling in the wake of the moving DNA fork (8). However, this mechanism is inconsistent with the results that the helicase domain cannot unwind DNA either in the holoenzyme of reverse gyrase or as a recombinant helicase domain moiety (7). A second proposed mechanism is that upon unwinding of the double-stranded DNA in a plasmid, reverse gyrase can separate the circular DNA into two independent topological domains, one with underwound base pairs and the other with positive supercoils. The unwound region will be renatured by reverse gyrase without affecting the positively supercoiled region, thus leaving behind positive supercoils (7, 26). It is not known how the enzyme switches from an unwinding activity to a renaturing one. The third proposed model is that reverse gyrase is capable of a direction-specific strand passage, thus allowing the accumulation of only positive DNA supercoils (9). The mechanistic basis for this direction-specific strand passage is unclear. Furthermore, it was also observed that at high enzyme concentrations, negatively supercoiled DNA could be generated in the presence of AMP-PNP (16). It was proposed that under such conditions the enzyme is capable of carrying out strand passage in both directions by either increasing or decreasing the linking numbers.



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  		FIG. 10.
Negative supercoiling by reverse gyrase/AMPPNP is resistant to relaxation by Tm topo I. Reactions of reverse gyrase (RG) with AMPPNP were carried at an enzyme/DNA ratio of 26 (lanes 2, 4, 6, 8, 10, and 12) or 9(lanes 3, 5, 7, 9, and 11). Half of the reactions contained 25 units of Tm topo I (lanes 4, 5, 8, 9, 12, and 13), and the other half did not. They were incubated under one of the following three conditions: 55 °C for 30 min (lanes 2–4), 80 °C for 30 min (lanes 6–9), and 80 °C for 30 min then shifted to 55 °C for another 30 min (lanes 10–13). Lanes 14 and 15 are for the reactions without reverse gyrase but with Tm topo I incubating at 55 and 80 °C, respectively. Lane 1 shows the substrate DNA. Under identical conditions without reverse gyrase, DNA can be completely relaxed by 1 unit of Tm topo I at 80 °C (data not shown). Notice that DNA supercoiling by reverse gyrase/AMPPNP at 80 °C was unchanged with an excess of Tm topo I.

 
The results shown in this paper suggest certain new aspects in the reverse gyrase mechanism. It is possible that the affinity of reverse gyrase for different conformations of DNA, such as single-stranded or double-stranded, is regulated by the binding of various adenine nucleotide cofactors. The switch from binding single-stranded to binding double-stranded DNA coupled with the strand passage activity from the topoisomerase domain results in a change in linking number. Because enzyme binding determines the magnitude of linking number change, the final extent of supercoiling change depends on enzyme/DNA ratio.

Reverse gyrase has a limited ability to unwind DNA (25), possibly reflecting its affinity for single-stranded DNA. Similar to other type IA topoisomerases, reverse gyrase may preferentially bind to the junction of single/double-stranded DNA (16, 27). The specificity for single-stranded DNA could be enhanced when ATP or AMPPNP is bound to the helicase domain of the enzyme. Although there is no structural data for the ternary complex of enzyme/DNA/ATP to support this hypothesis, the structure of a ternary complex of a bacterial helicase PcrA demonstrates the binding of a single-stranded DNA segment with the ATP-bound enzyme (28). This unwinding can be greatly facilitated by the high temperature at which the reaction is carried out. The extensive melting of double-stranded DNA results in the accumulation of positive supercoils. With the non-hydrolyzable analog AMPPNP, the enzyme forms a stable ternary complex. The topoisomerase moieties of the enzymes bound near the junction of double/single-stranded DNA allow a gradual relaxation of the positive supercoils, thus generating negative supercoils after the bound enzymes are removed from DNA. Because the linking deficiency (negative supercoiling) is a result of the binding of reverse gyrase to the single-stranded region, the negative supercoiling will be refractory to topo I relaxation, a conclusion supported by our experiments. This proposed mechanism would also require reverse gyrase to be able to remove positive supercoils, an activity that type IA topoisomerases usually do not possess. However, at the double/single-strand junction, previous data showed that a type IA enzyme like bacterial topo I or eucaryotic topo III can indeed relax positive DNA supercoils (27, 29, 30). We have also shown that whereas reverse gyrase cannot relax positive supercoils from a plasmid substrate, it is capable of removing them from a DNA substrate containing a single-stranded bubble.2



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  		FIG. 11.
Diagrammatic representation of a proposed mechanism for the positive supercoiling by reverse gyrase (Rev-Gyrase). 1, starting with negatively supercoiled plasmid DNA, reverse gyrase in the presence of a cofactor can readily relax the DNA, an activity shared by most type IA topoisomerases. 2, binding of reverse gyrase complexed with ATP to the single-stranded DNA results in an unwound region and compensatory positive DNA supercoiling. The enzyme bound to the single-stranded region is not shown here. 3, the hydrolysis of ATP to ADP results in changing the binding affinity to double-stranded DNA, which coupled with a strand passage activity from the topoisomerase domain, leads to the rewinding of the denatured bubble. The annealing of the single-stranded DNA greatly reduces the topoisomerase activity, thus promoting the retention of positive supercoils. 4, after the removal of proteins, the reaction product is positively supercoiled.

 
Starting with negatively supercoiled DNA, the topoisomerase activity in reverse gyrase can readily relax DNA in the presence of either ADP or ATP (see the diagrammatic representation in Fig 11). Such negative supercoil relaxation activity is innate to all type IA topoisomerases. With relaxed DNA as a substrate, reverse gyrase can change the DNA supercoiling through its cofactor-dependent binding affinity for either single- or double-stranded DNA. When reverse gyrase is bound to ATP, its affinity for the single-stranded region will result in an extensive unwound segment of DNA, the length of which depends on the stoichiometry of enzyme/DNA and reaction temperature. The unwinding of the duplex also leads to a compensatory positive supercoiling in the molecule. Subsequent hydrolysis of ATP results in the enzyme complexed with ADP, which we propose has a higher affinity for double-stranded DNA over single-stranded. The annealing of DNA strands coupled with the strand passage activity in the topoisomerase I domain allows the positive supercoils to be retained after removal of bound enzymes. Why is the enzyme incapable of relaxing the positive supercoils here? In this mode of action the positive supercoil relaxation activity from the enzymes bound at the junction of double/single-stranded DNA is diminished due to the hydrolysis of ATP into ADP. With the ADP-bound form, the enzyme reanneals the single-stranded region and eliminates the DNA bubble, thus preventing the topoisomerase domain from removing the positive supercoils. This proposed mechanism can also account for the low level of positive supercoiling observed in reverse gyrase reactions with ADP. The ADP-bound enzyme can stabilize the double-stranded region against the thermal unwinding and denaturation. In the absence of the single-stranded binding activity of the ATP-bound form, the unwound segment will be limited to pre-melting regions, accounting for a lower level of positive supercoiling introduced by reverse gyrase with ADP.

The data reported in this paper support two key elements in the proposed mechanism. First is the cofactor dependence of supercoiling activity, which is possibly due to a differential affinity of reverse gyrase for double-versus single-stranded DNA. Although the helicase domain in the enzyme retains the DNA-dependent ATPase activity, it does not have the translocation activity and cannot displace DNA from its complementary strand (7). However, it may still have the ability to discriminate between single-stranded and double-stranded DNA depending upon the bound cofactor. Second is the stoichiometry-dependent DNA supercoiling, suggesting the extent of enzyme binding to DNA determines the level of DNA supercoiling. This property is different from all other topoisomerases studied thus far, including DNA gyrase. The stabilizing function of reverse gyrase due to its association with DNA may have relevance to its in vivo functions in maintaining genome stability in a high temperature growth environment. Recent data suggest that reverse gyrase may have a function to stabilize the genome against DNA cleavage due to high temperature through its binding to damaged DNA sites (12). The biochemical basis for the reverse gyrase to discriminate and associate with different DNA structures will be an interesting area for further investigation.


   FOOTNOTES
 
* This work was supported by National Institutes of Heath Grant GM29006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 919-684-6501; Fax: 19-684-8885; E-mail: hsieh{at}biochem.duke.edu.

1 The abbreviations used are: topo, topoisomerase; AMPPNP, 5'-adenylyl-{beta},{gamma}-imidodiphosphate; Tm, T. maritima. Back

2 T.-s. Hsieh and J. L. Plank, unpublished data. Back


   ACKNOWLEDGMENTS
 
We appreciate helpful discussions from Jode Plank, Tammy Collins, and Sabrina Rozenman and the able technical assistance from Larry Li. We thank Dr. Chapin Rodriguez for a kind gift of the expression vector of reverse gyrase.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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