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DNA topology: Topoisomerases keep it simple
R778
Dispatch
DNA topology: Topoisomerases keep it simple Andrew D. Bates* and Anthony Maxwell†
The ability of type II DNA topoisomerases to perturb the equilibrium distributions of DNA topoisomers is a consequence of their ability to hydrolyse ATP. A sliding mechanism of topoisomerase action has been proposed to account for this phenomenon. Addresses: *School of Biological Sciences, University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, UK. E-mail: [email protected]. †Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK. E-mail: [email protected]. Current Biology 1997, 7:R778–R781 http://biomednet.com/elecref/09609822007R0778 © Current Biology Ltd ISSN 0960-9822
One of the basic tenets of enzyme-catalysed reactions is that an enzyme cannot alter the equilibrium between substrate and product — it can only accelerate the rate of interconversion between them. However, if the enzyme couples an energy-yielding reaction, such as the hydrolysis of ATP, to the conversion of substrate to product, then an apparently non-equilibrium mixture can be generated. It has long been a puzzle why many type II DNA topoisomerases hydrolyse ATP while catalysing energetically favourable reactions, such as the relaxation of supercoiled DNA. Recent experiments by Rybenkov et al. [1] show that these enzymes are able to reduce the topological complexity of DNA relative to equilibrium values, and ATP hydrolysis is invoked to explain their ability to perturb the thermodynamic equilibrium. DNA topoisomerases catalyse the interconversion of different topological forms of DNA, including relaxed and supercoiled, knotted and unknotted, and catenated (linked) and unlinked DNA circles (Figure 1). They are classified into two types: type I enzymes catalyse reactions involving transient single-strand breaks in DNA, and type II enzymes catalyse reactions involving transient double-strand breaks [2]. The current mechanistic model for type II enzymes involves the binding of two segments of DNA: a G (gate) segment, which is broken in both strands by the enzyme with the formation of covalent bonds between active-site tyrosines and 5′ phosphates in the DNA, and a T (transported) segment, which is captured by an ATP-operated clamp and passed through the enzyme-stabilised break in the G segment [3]. This passage of one double-stranded segment through another, in either an intramolecular or intermolecular fashion, can accomplish all the reactions illustrated in Figure 1. Most reactions catalysed by type II enzymes require ATP hydrolysis. In the case of the bacterial enzyme
DNA gyrase, which is the only type II enzyme known to be capable of introducing supercoils into DNA, the requirement for ATP is clear. The introduction of supercoils into a relaxed DNA circle is energetically unfavourable and it seems that, under the right conditions, much of the energy of ATP hydrolysis can be converted into DNA supercoiling energy [4–6]. Gyrase performs this directional intramolecular reaction by wrapping a segment of DNA around itself, and delivering a nearby T segment to the ATP-operated clamp with a specific orientation [7]. In the case of the eukaryotic type II enzymes or the bacterial enzyme topoisomerase IV (topo IV), which are specifically implicated in the unlinking of daughter chromosomes after DNA replication [8,9], the energy requirement is not so clear. These enzymes do not wrap DNA, and they catalyse reactions that appear to be energetically favourable, such as the relaxation of supercoiled DNA or the unlinking of catenanes, yet they too require the hydrolysis of ATP. It has been suggested that this energy input is needed to drive the enzyme through an ordered series of conformational changes [10], in other words, to facilitate the opening and closing
Figure 1
(a)
(b) +
(c)
Current Biology
The reactions carried out by type II topoisomerases: (a) supercoiling/ relaxation; (b) catenation/decatenation; (c) knotting/unknotting. All these transformations can be performed by the passage of one doublestranded DNA segment through another (double-headed arrows).
Dispatch
Figure 2
R779
using an analogous method, and similarly found to be far from the equilibrium value when enzyme and ATP were present: 90-fold lower for pAB4, and 50-fold lower for P4.
Topo IV + ATP
Equilibrated mixture
Topo IV + ATP
Topo IV + ATP
Steady-state mixture (Topo IV + ATP) Current Biology
A schematic illustration of the reaction of topoisomerase IV with DNA catenanes. The steady-state mixture of catenated and unlinked products formed in the presence of topo IV and ATP contains a lower proportion of catenanes than the equilibrium mixture formed by the cyclisation of P4 DNA in the presence of pAB4 plasmid. Catenanes are in red, and unlinked DNA circles are in green.
of the ATP-operated clamp that captures the T segment and initiates the strand-passage process. A number of recent studies have suggested that these enzymes may be acting more specifically than previously thought — for example topo IV has been shown to be more active in decatenation than in relaxation reactions [11,12]. Recent work by Rybenkov et al. [1] now suggests an alternative explanation for the ATP requirement of these enzymes. In previous studies, these workers developed a system that allows equilibration of catenanes without the requirement for enzymic catalysis, consisting of a nicked plasmid (pAB4) and linear phage P4 DNA, which has long cohesive ends [13]. Under conditions of equilibration, the P4 DNA molecules circularise, trapping some of the pAB4 plasmids to form dimeric catenanes. The proportion of catenated DNA at equilibrium can be determined using an agarose gel. This system enabled Rybenkov et al. [1] to compare samples of catenated DNA generated either by equilibration or by the action of type II topoisomerases (Figure 2). The fractions of catenated DNA formed were shown to be very different in these two cases. When a sample with twice the equilibrium concentration of catenanes was treated with topo IV and ATP at a range of enzyme:DNA ratios, the steady-state fraction of catenanes in the sample was as much as 16-fold lower than the value at equilibrium in the absence of enzyme, and this same fraction was obtained when the initial substrate was a mixture of unlinked circles at the same concentrations (Figure 2). For both pAB4 and P4 DNA, the fraction of knotted versus unknotted species was also measured
With supercoiled pAB4 plasmid, the relaxation reaction was studied and topo IV was found to generate a distribution of topoisomers with a narrower range of linking numbers (Lk) than that generated by wheat-germ topoisomerase I, an ATP-independent DNA relaxing enzyme. This means that topo IV can reduce the level of supercoiling in a sample below the equilibrium level established by thermal energy [14]. Similar experiments were carried out with other type II enzymes from phage T2, Saccharomyces cerevisiae, Drosophila melanogaster and human cells, and although the magnitude of the effect varied from enzyme to enzyme, all reduced the fraction of catenanes and knots and narrowed the Lk distribution. In other words, all these enzymes actively simplify DNA topology relative to the complexity expected at equilibrium. To comply with the laws of thermodynamics, the energy of ATP hydrolysis must be invoked to drive these reactions away from their equilibrium positions. With hindsight, it seems clear that coupled ATP hydrolysis should enable type II topoisomerases to generate non-equilibrated products, given the persuasive example of the supercoiling reaction catalysed by DNA gyrase (see above). The extreme nature of the gyrase case may have blinded us to the rather more subtle effects of the other type II enzymes. The experiments of Rybenkov et al. [1] elegantly demonstrate these effects, and show that all type II enzymes act with a specific directionality: gyrase in the direction of increasing supercoiling complexity, and the other enzymes in the direction of simplifying DNA topology. This behaviour is consistent with our understanding of the roles of these enzymes in vivo; gyrase maintains the level of negative supercoiling in bacterial cells and specifically removes positive supercoiling generated by transcription and replication, whereas the other type II enzymes relax both positive and negative supercoiling, and unlink daughter chromosomes during and after replication [15]. To envisage a mechanism that will explain this behaviour is a more difficult matter. In order to perturb the equilibrium between catenated and unlinked DNA, for example, the enzymes need to identify specifically a G segment and a T segment that are located on two catenated circles in preference to a pair of segments on unlinked molecules. Rybenkov et al. [1] propose a model in which each enzyme can bind to three sites on DNA. Initial binding to two segments of a DNA circle, the G segment (which will become cleaved) and another distant site, might constrain a catenated or knotted strand or a supercoiled region (destined to become the T segment) within a shorter loop of the circle, and thus make capture of the T segment more probable (Figure 3). Although there is some evidence for
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the interaction of yeast topoisomerase II with two distant sites on DNA [16], such an effect will not, of itself, explain the perturbation of the equilibrium. It is therefore envisaged that the enzyme can slide along the DNA at the third site, constraining the prospective T segment in a progressively smaller region and making its capture increasingly probable (Figure 3). This directional sliding would have to be the energy-requiring step of the process. This idea, however, is at odds with what we know of the properties of the ATP binding and hydrolysing domains of these enzymes. There is abundant evidence from studies of gyrase and yeast topoisomerase II that binding of ATP acts to close a clamp by domain dimerisation, trapping the T segment, which is then passed through the G segment [3,17,18]. Subsequent hydrolysis of the ATP re-opens the clamp ready for a new reaction. It is difficult to imagine a dual role for ATP hydrolysis both in clamp opening and closing, and in driving the sliding of the topoisomerase on DNA, but as there is no obvious alternative mechanism to account for these phenomena, we are left with this intriguing model. The work of Rybenkov et al. [1] also raises other interesting questions. For example, which part of the enzyme binds the postulated third DNA segment? It would have been tempting to propose that this segment binds to the carboxy-terminal domain, which does not have an identified role in the reaction mechanism of non-gyrase type II enzymes. However, Rybenkov et al. [1] show that a mutant form of yeast topoisomerase II that lacks the last approximately 200 amino acids can also perturb the topological equilibria (actually in a more extreme fashion than the full-length enzyme). Does DNA gyrase have the same ability to generate nonequilibrium topoisomer products? Although the answer is obviously ‘yes’ in the case of the supercoiling reaction, it would be interesting to compare the products of the ATPdependent and ATP-independent relaxation reactions of the truncated form of the enzyme [19], and to examine the (albeit rather inefficient) decatenation reaction of fulllength gyrase. What is the free-energy requirement of the proposed sliding reaction? Is the extent of perturbation of the equilibria dependent on the amount of free energy available from ATP hydrolysis? It would be interesting to examine the distribution of the products of type II topoisomerase reactions in the presence of ATP analogues that yield more or less free energy of hydrolysis than ATP, or using a range of ATP:ADP ratios [4]. Perhaps the most important aspect of the work of Rybenkov et al. [1] is that it has clearly established that all the type II topoisomerases can perturb the equilibria of
Figure 3
G segment T segment
Topoisomerase ATP hydrolysis
Current Biology
The model proposed by Rybenkov et al. [1] for the ATP-driven decatenation reaction of type II topoisomerases. The enzyme (blue) binds to the G segment and another distant site, at which ATP hydrolysis can drive the enzyme along the DNA, constraining the T segment in a smaller loop and making its capture more probable. The notch in the enzyme indicates the ATP-operated clamp and the arrows indicate the ATP-dependent movement of DNA.
DNA topological forms in the direction consistent with their biological role. How the enzymes achieve this remains to be shown. It is clear that there are many more complexities of these remarkable enzymes which remain to be unravelled. Acknowledgements We would like to thank Tim Hammonds, Sotirios Kampranis, Stephen Robertson and Melisa Wall for helpful discussions and comments on the manuscript. AM is a Lister-Jenner Research Fellow.
References 1. Rybenkov VV, Ullsperger C, Vologodskii AV, Cozzarelli NR: Simplification of DNA topology below equilibrium values by type II topoisomerases. Science 1997, 277:690-693. 2. Bates AD, Maxwell A: DNA Topology. Edited by Rickwood D, Male D Oxford: IRL Press; 1993. 3. Roca J, Wang JC: The capture of a DNA double-helix by an ATPdependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. Cell 1992, 71:833-840. 4. Westerhoff HV, O’Dea MH, Maxwell A, Gellert M: DNA supercoiling by DNA gyrase. A static head analysis. Cell Biophys 1988, 12:157181. 5. Bates AD, Maxwell A: DNA gyrase can supercoil DNA circles as small as 174 base pairs. EMBO J 1989, 8:1861-1866. 6. Cullis PM, Maxwell A, Weiner DP: Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling. Biochemistry 1992, 31:9642-9646. 7. Bates AD, O’Dea MH, Gellert M: Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage and DNA supercoiling. Biochemistry 1996, 35:1408-1416. 8. DiNardo S, Voelkel K, Sternglanz R: DNA topoisomerase II mutant of Saccharomyces cerevisiae: Topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci USA 1984, 81:2616-2620.
Dispatch
9. Adams DE, Shekhtman EM, Zechiedrich EL, Schmid MB, Cozzarelli NR: The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Cell 1992, 71:277-288. 10. Liu LF, Liu C-C, Alberts BM: Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell 1980, 19:697-707. 11. Peng H, Marians KJ: Escherichia coli topoisomerase IV. Purification, characterisation, subunit structure and subunit interactions. J Biol Chem 1993, 268:24481-24490. 12. Ullsperger C, Cozzarelli NR: Contrasting enzymatic activities of topoisomerase IV and DNA gyrase from Escherichia coli. J Biol Chem 1996, 271:31549-31555. 13. Rybenkov VV, Vologodskii AV, Cozzarelli NR: The effect of ionic conditions on the conformations of supercoiled DNA. II. Equilibrium catenation. J Mol Biol 1997, 267:312-323. 14. Depew RE, Wang JC: Conformational fluctuations of DNA helix. Proc Natl Acad Sci USA 1975, 72:4275-4279. 15. Wang JC: DNA topoisomerases: why so many? J Biol Chem 1991, 266:6659-6662. 16. Roca J, Berger JM, Wang JC: On the simultaneous binding of eukaryotic DNA topoisomerase II to a pair of double-stranded DNA helices. J Biol Chem 1993, 268:14250-14255. 17. Wigley DB, Davies GJ, Dodson EJ, Maxwell A, Dodson G: Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature 1991, 351:624-629. 18. Ali JA, Jackson AP, Howells AJ, Maxwell A: The 43 kilodalton Nterminal fragment of the DNA gyrase B protein hydrolyses ATP and binds coumarin drugs. Biochemistry 1993, 32:2717-2724. 19. Kampranis SC, Maxwell A: Conversion of DNA gyrase into a conventional type II topoisomerase. Proc Natl Acad Sci USA 1996, 93:14416-14421.
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Dispatch
DNA topology: Topoisomerases keep it simple Andrew D. Bates* and Anthony Maxwell†
The ability of type II DNA topoisomerases to perturb the equilibrium distributions of DNA topoisomers is a consequence of their ability to hydrolyse ATP. A sliding mechanism of topoisomerase action has been proposed to account for this phenomenon. Addresses: *School of Biological Sciences, University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, UK. E-mail: [email protected]. †Department of Biochemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK. E-mail: [email protected]. Current Biology 1997, 7:R778–R781 http://biomednet.com/elecref/09609822007R0778 © Current Biology Ltd ISSN 0960-9822
One of the basic tenets of enzyme-catalysed reactions is that an enzyme cannot alter the equilibrium between substrate and product — it can only accelerate the rate of interconversion between them. However, if the enzyme couples an energy-yielding reaction, such as the hydrolysis of ATP, to the conversion of substrate to product, then an apparently non-equilibrium mixture can be generated. It has long been a puzzle why many type II DNA topoisomerases hydrolyse ATP while catalysing energetically favourable reactions, such as the relaxation of supercoiled DNA. Recent experiments by Rybenkov et al. [1] show that these enzymes are able to reduce the topological complexity of DNA relative to equilibrium values, and ATP hydrolysis is invoked to explain their ability to perturb the thermodynamic equilibrium. DNA topoisomerases catalyse the interconversion of different topological forms of DNA, including relaxed and supercoiled, knotted and unknotted, and catenated (linked) and unlinked DNA circles (Figure 1). They are classified into two types: type I enzymes catalyse reactions involving transient single-strand breaks in DNA, and type II enzymes catalyse reactions involving transient double-strand breaks [2]. The current mechanistic model for type II enzymes involves the binding of two segments of DNA: a G (gate) segment, which is broken in both strands by the enzyme with the formation of covalent bonds between active-site tyrosines and 5′ phosphates in the DNA, and a T (transported) segment, which is captured by an ATP-operated clamp and passed through the enzyme-stabilised break in the G segment [3]. This passage of one double-stranded segment through another, in either an intramolecular or intermolecular fashion, can accomplish all the reactions illustrated in Figure 1. Most reactions catalysed by type II enzymes require ATP hydrolysis. In the case of the bacterial enzyme
DNA gyrase, which is the only type II enzyme known to be capable of introducing supercoils into DNA, the requirement for ATP is clear. The introduction of supercoils into a relaxed DNA circle is energetically unfavourable and it seems that, under the right conditions, much of the energy of ATP hydrolysis can be converted into DNA supercoiling energy [4–6]. Gyrase performs this directional intramolecular reaction by wrapping a segment of DNA around itself, and delivering a nearby T segment to the ATP-operated clamp with a specific orientation [7]. In the case of the eukaryotic type II enzymes or the bacterial enzyme topoisomerase IV (topo IV), which are specifically implicated in the unlinking of daughter chromosomes after DNA replication [8,9], the energy requirement is not so clear. These enzymes do not wrap DNA, and they catalyse reactions that appear to be energetically favourable, such as the relaxation of supercoiled DNA or the unlinking of catenanes, yet they too require the hydrolysis of ATP. It has been suggested that this energy input is needed to drive the enzyme through an ordered series of conformational changes [10], in other words, to facilitate the opening and closing
Figure 1
(a)
(b) +
(c)
Current Biology
The reactions carried out by type II topoisomerases: (a) supercoiling/ relaxation; (b) catenation/decatenation; (c) knotting/unknotting. All these transformations can be performed by the passage of one doublestranded DNA segment through another (double-headed arrows).
Dispatch
Figure 2
R779
using an analogous method, and similarly found to be far from the equilibrium value when enzyme and ATP were present: 90-fold lower for pAB4, and 50-fold lower for P4.
Topo IV + ATP
Equilibrated mixture
Topo IV + ATP
Topo IV + ATP
Steady-state mixture (Topo IV + ATP) Current Biology
A schematic illustration of the reaction of topoisomerase IV with DNA catenanes. The steady-state mixture of catenated and unlinked products formed in the presence of topo IV and ATP contains a lower proportion of catenanes than the equilibrium mixture formed by the cyclisation of P4 DNA in the presence of pAB4 plasmid. Catenanes are in red, and unlinked DNA circles are in green.
of the ATP-operated clamp that captures the T segment and initiates the strand-passage process. A number of recent studies have suggested that these enzymes may be acting more specifically than previously thought — for example topo IV has been shown to be more active in decatenation than in relaxation reactions [11,12]. Recent work by Rybenkov et al. [1] now suggests an alternative explanation for the ATP requirement of these enzymes. In previous studies, these workers developed a system that allows equilibration of catenanes without the requirement for enzymic catalysis, consisting of a nicked plasmid (pAB4) and linear phage P4 DNA, which has long cohesive ends [13]. Under conditions of equilibration, the P4 DNA molecules circularise, trapping some of the pAB4 plasmids to form dimeric catenanes. The proportion of catenated DNA at equilibrium can be determined using an agarose gel. This system enabled Rybenkov et al. [1] to compare samples of catenated DNA generated either by equilibration or by the action of type II topoisomerases (Figure 2). The fractions of catenated DNA formed were shown to be very different in these two cases. When a sample with twice the equilibrium concentration of catenanes was treated with topo IV and ATP at a range of enzyme:DNA ratios, the steady-state fraction of catenanes in the sample was as much as 16-fold lower than the value at equilibrium in the absence of enzyme, and this same fraction was obtained when the initial substrate was a mixture of unlinked circles at the same concentrations (Figure 2). For both pAB4 and P4 DNA, the fraction of knotted versus unknotted species was also measured
With supercoiled pAB4 plasmid, the relaxation reaction was studied and topo IV was found to generate a distribution of topoisomers with a narrower range of linking numbers (Lk) than that generated by wheat-germ topoisomerase I, an ATP-independent DNA relaxing enzyme. This means that topo IV can reduce the level of supercoiling in a sample below the equilibrium level established by thermal energy [14]. Similar experiments were carried out with other type II enzymes from phage T2, Saccharomyces cerevisiae, Drosophila melanogaster and human cells, and although the magnitude of the effect varied from enzyme to enzyme, all reduced the fraction of catenanes and knots and narrowed the Lk distribution. In other words, all these enzymes actively simplify DNA topology relative to the complexity expected at equilibrium. To comply with the laws of thermodynamics, the energy of ATP hydrolysis must be invoked to drive these reactions away from their equilibrium positions. With hindsight, it seems clear that coupled ATP hydrolysis should enable type II topoisomerases to generate non-equilibrated products, given the persuasive example of the supercoiling reaction catalysed by DNA gyrase (see above). The extreme nature of the gyrase case may have blinded us to the rather more subtle effects of the other type II enzymes. The experiments of Rybenkov et al. [1] elegantly demonstrate these effects, and show that all type II enzymes act with a specific directionality: gyrase in the direction of increasing supercoiling complexity, and the other enzymes in the direction of simplifying DNA topology. This behaviour is consistent with our understanding of the roles of these enzymes in vivo; gyrase maintains the level of negative supercoiling in bacterial cells and specifically removes positive supercoiling generated by transcription and replication, whereas the other type II enzymes relax both positive and negative supercoiling, and unlink daughter chromosomes during and after replication [15]. To envisage a mechanism that will explain this behaviour is a more difficult matter. In order to perturb the equilibrium between catenated and unlinked DNA, for example, the enzymes need to identify specifically a G segment and a T segment that are located on two catenated circles in preference to a pair of segments on unlinked molecules. Rybenkov et al. [1] propose a model in which each enzyme can bind to three sites on DNA. Initial binding to two segments of a DNA circle, the G segment (which will become cleaved) and another distant site, might constrain a catenated or knotted strand or a supercoiled region (destined to become the T segment) within a shorter loop of the circle, and thus make capture of the T segment more probable (Figure 3). Although there is some evidence for
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Current Biology, Vol 7 No 12
the interaction of yeast topoisomerase II with two distant sites on DNA [16], such an effect will not, of itself, explain the perturbation of the equilibrium. It is therefore envisaged that the enzyme can slide along the DNA at the third site, constraining the prospective T segment in a progressively smaller region and making its capture increasingly probable (Figure 3). This directional sliding would have to be the energy-requiring step of the process. This idea, however, is at odds with what we know of the properties of the ATP binding and hydrolysing domains of these enzymes. There is abundant evidence from studies of gyrase and yeast topoisomerase II that binding of ATP acts to close a clamp by domain dimerisation, trapping the T segment, which is then passed through the G segment [3,17,18]. Subsequent hydrolysis of the ATP re-opens the clamp ready for a new reaction. It is difficult to imagine a dual role for ATP hydrolysis both in clamp opening and closing, and in driving the sliding of the topoisomerase on DNA, but as there is no obvious alternative mechanism to account for these phenomena, we are left with this intriguing model. The work of Rybenkov et al. [1] also raises other interesting questions. For example, which part of the enzyme binds the postulated third DNA segment? It would have been tempting to propose that this segment binds to the carboxy-terminal domain, which does not have an identified role in the reaction mechanism of non-gyrase type II enzymes. However, Rybenkov et al. [1] show that a mutant form of yeast topoisomerase II that lacks the last approximately 200 amino acids can also perturb the topological equilibria (actually in a more extreme fashion than the full-length enzyme). Does DNA gyrase have the same ability to generate nonequilibrium topoisomer products? Although the answer is obviously ‘yes’ in the case of the supercoiling reaction, it would be interesting to compare the products of the ATPdependent and ATP-independent relaxation reactions of the truncated form of the enzyme [19], and to examine the (albeit rather inefficient) decatenation reaction of fulllength gyrase. What is the free-energy requirement of the proposed sliding reaction? Is the extent of perturbation of the equilibria dependent on the amount of free energy available from ATP hydrolysis? It would be interesting to examine the distribution of the products of type II topoisomerase reactions in the presence of ATP analogues that yield more or less free energy of hydrolysis than ATP, or using a range of ATP:ADP ratios [4]. Perhaps the most important aspect of the work of Rybenkov et al. [1] is that it has clearly established that all the type II topoisomerases can perturb the equilibria of
Figure 3
G segment T segment
Topoisomerase ATP hydrolysis
Current Biology
The model proposed by Rybenkov et al. [1] for the ATP-driven decatenation reaction of type II topoisomerases. The enzyme (blue) binds to the G segment and another distant site, at which ATP hydrolysis can drive the enzyme along the DNA, constraining the T segment in a smaller loop and making its capture more probable. The notch in the enzyme indicates the ATP-operated clamp and the arrows indicate the ATP-dependent movement of DNA.
DNA topological forms in the direction consistent with their biological role. How the enzymes achieve this remains to be shown. It is clear that there are many more complexities of these remarkable enzymes which remain to be unravelled. Acknowledgements We would like to thank Tim Hammonds, Sotirios Kampranis, Stephen Robertson and Melisa Wall for helpful discussions and comments on the manuscript. AM is a Lister-Jenner Research Fellow.
References 1. Rybenkov VV, Ullsperger C, Vologodskii AV, Cozzarelli NR: Simplification of DNA topology below equilibrium values by type II topoisomerases. Science 1997, 277:690-693. 2. Bates AD, Maxwell A: DNA Topology. Edited by Rickwood D, Male D Oxford: IRL Press; 1993. 3. Roca J, Wang JC: The capture of a DNA double-helix by an ATPdependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. Cell 1992, 71:833-840. 4. Westerhoff HV, O’Dea MH, Maxwell A, Gellert M: DNA supercoiling by DNA gyrase. A static head analysis. Cell Biophys 1988, 12:157181. 5. Bates AD, Maxwell A: DNA gyrase can supercoil DNA circles as small as 174 base pairs. EMBO J 1989, 8:1861-1866. 6. Cullis PM, Maxwell A, Weiner DP: Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling. Biochemistry 1992, 31:9642-9646. 7. Bates AD, O’Dea MH, Gellert M: Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage and DNA supercoiling. Biochemistry 1996, 35:1408-1416. 8. DiNardo S, Voelkel K, Sternglanz R: DNA topoisomerase II mutant of Saccharomyces cerevisiae: Topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci USA 1984, 81:2616-2620.
Dispatch
9. Adams DE, Shekhtman EM, Zechiedrich EL, Schmid MB, Cozzarelli NR: The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Cell 1992, 71:277-288. 10. Liu LF, Liu C-C, Alberts BM: Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell 1980, 19:697-707. 11. Peng H, Marians KJ: Escherichia coli topoisomerase IV. Purification, characterisation, subunit structure and subunit interactions. J Biol Chem 1993, 268:24481-24490. 12. Ullsperger C, Cozzarelli NR: Contrasting enzymatic activities of topoisomerase IV and DNA gyrase from Escherichia coli. J Biol Chem 1996, 271:31549-31555. 13. Rybenkov VV, Vologodskii AV, Cozzarelli NR: The effect of ionic conditions on the conformations of supercoiled DNA. II. Equilibrium catenation. J Mol Biol 1997, 267:312-323. 14. Depew RE, Wang JC: Conformational fluctuations of DNA helix. Proc Natl Acad Sci USA 1975, 72:4275-4279. 15. Wang JC: DNA topoisomerases: why so many? J Biol Chem 1991, 266:6659-6662. 16. Roca J, Berger JM, Wang JC: On the simultaneous binding of eukaryotic DNA topoisomerase II to a pair of double-stranded DNA helices. J Biol Chem 1993, 268:14250-14255. 17. Wigley DB, Davies GJ, Dodson EJ, Maxwell A, Dodson G: Crystal structure of an N-terminal fragment of the DNA gyrase B protein. Nature 1991, 351:624-629. 18. Ali JA, Jackson AP, Howells AJ, Maxwell A: The 43 kilodalton Nterminal fragment of the DNA gyrase B protein hydrolyses ATP and binds coumarin drugs. Biochemistry 1993, 32:2717-2724. 19. Kampranis SC, Maxwell A: Conversion of DNA gyrase into a conventional type II topoisomerase. Proc Natl Acad Sci USA 1996, 93:14416-14421.
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