Quarterly Reviews of Biophysics , (), pp. –. Printed in the United Kingdom # Cambridge University Press Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine
JAMES C. WANG Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts , USA
. . The double helix structure of DNA and its topological ramifications . Entering the DNA topoisomerases . Type II DNA topoisomerases . . Transporting one DNA double helix through another . The clamp model: a type II DNA topoisomerase as an ATP-modulated protein clamp . The number of gates in the protein clamp . Three-dimensional structures of type II DNA topoisomerase fragments . How are the various subfragments connected? . A molecular model of DNA transport by a type II DNA topoisomerase . . ATP utilization . How tight is the coupling between DNA transport and ATP binding and hydrolysis? .. Dependence of the coupling efficiency on the degree of supercoiling of the DNA substrate .. Coupling efficiency and DNA binding . DNA relaxation by a type II DNA topoisomerase: how is the high efficiency of coupling achieved? .. ATP hydrolysis and the closure of the enzyme clamp .. Coupling of ATPase activity to DNA binding .. Structural changes in the enzyme upon closure of the N-gate .. ATP binding\hydrolysis and DNA cleavage .. Entrapment of the T-segment and opening of the DNA gate .. Closure of the DNA gate and exit of the T-segment J. C. Wang . Directionality of DNA transport: why is bacterial gyrase unique? .. Structural basis of the ability of bacterial DNA gyrase to catalyse the ATP-dependent negative supercoiling of DNA .. The ATP-independent DNA relaxation activity of gyrase . . .
. .. The double helix structure of DNA and its topological ramifications The discovery of the double helix structure of DNA led immediately to questions on the mechanics of unravelling its intertwined strands during replication. If a parental DNA is to be duplicated into two progeny molecules by separating its two strands and copying each, then the strands must untwine rapidly during replication (Watson & Crick, ). That DNA indeed replicates in such a semiconservative fashion was soon demonstrated by the Meselson–Stahl experiment (). At first, it appeared that the unravelling of the intertwined strands should not pose an insurmountable mechanical problem. The two strands at one end of a linear DNA, for example, can be pulled apart with concomitant rotation of the double-stranded portion of the molecule around its helical axis. If the strands of a DNA double helix are to separate at an estimated replication rate of base pairs (bp) per minute, then the speed of this rotation would be revolutions per minute from the bp per turn helical geometry of the double helix. This speed, though impressive, seemed reasonable: owing to the slender rod-like shape of the double helix, the estimated viscous drag for this rotational motion is actually rather modest (Meselson, ). Two findings in the s, however, heightened interests in the DNA uncoiling problem. First, evidence began to accumulate that the lengths of DNA molecules from natural sources are much longer than previously realized. Once it was found that mechanical shear during DNA preparation can cause extensive breakage of DNA, new records of longer and longer DNA molecules were set. It soon became clear that an entire chromosome containing tens of millions of base pairs is consisted of a single DNA molecule, with a length of several millimetres along its contour (Kavenoff et al. ). For such a long DNA, the simple idea of unravelling its intertwined strands by rotating the entire thread-like molecule around its helical axis is untenable. Second, the s also saw the discovery of various ring-shaped or ‘circular’ DNA molecules. Of particular importance was the finding that the small DNA of polyoma virus is a double-stranded ring with intact strands (Dulbecco & Vogt, ; Weil & Vinograd, ). It was shown that for such a ‘covalently closed’ DNA, the two complementary strands are topologically linked, as predicted by the double-helix structure: a pair of intertwined single-stranded DNA rings in a double-stranded DNA ring can not A simple molecular machine
(a) (b)
A
B
C
Fig. . Schematic drawings illustrating two topological problems of DNA during semiconservative replication. In (a), the general problem of separating the two parental strands is illustrated for a double-stranded DNA ring. In this two-line representation of the duplex DNA ring, each of the circular DNA strands is represented by a closed line. Because of the double helix geometry of DNA, the two strands are topologically linked; the degree of linkage between them must gradually reduce as DNA replication proceeds, and the two strands must become completely unlinked at the end of a round of replication. For the two closed lines drawn in the illustration, one way of unlinking them is to carry out multiple cycles of a simple operation: in each cycle, one line is transiently broken, the other line is then passed through the break once before the break is resealed. The linking number (Lk) between the two closed strands is the minimal number of such cycles that is required to unlink them. Inspection of the particular drawing shows that all crossovers are of the same sign and each strand passage event removes two crossovers; Lk is thus equal to one half of the number of crossovers. For other ways of defining Lk, see Crick (), Cozzarelli et al. (), Wang (). In (b), the conversion of twists between the strands of an unreplicated DNA segment to intertwines between two fully replicated progeny DNA molecules is illustrated. Two replication forks are shown to approach each other near the end of a round of DNA replication (drawing A). If copying of the unreplicated portion of the parental DNA segment is faster than unlinking the parental strands coiled around each other, two intertwined progeny molecules are formed (C). Under such conditions, the replication product of a circular DNA would be a pair of multiply linked rings. Illustration taken from Varshavsky et al.(). be separated without breaking at least one of the strands. A parameter defining the degree of topological linkage between the two strands of a duplex DNA ring is termed the linking number (Lk). As this quantity will pop up again in subsequent discussions, an operational definition of Lk is illustrated in Fig. a. Whereas the problem of unravelling the complementary strands of a long linear DNA during replication might appear to be kinematic in nature, the same problem for a covalently closed DNA ring is surely a topological one. Actually, because a linear chromosome inside a cell is organized in a compact form with J. C. Wang multiple loops, the problem of separating its strands is not all that different from that for a covalently closed DNA ring. Therefore, it would appear that during evolution, as DNA became very long or circular, a solution must be found for the disentanglement of its topologically intertwined strands during replication. The separation of two parental DNA strands during replication is the best known topological manifestation of the double helix structure. It is not the only one, however, and a variation of the theme is illustrated in Fig. b. In the top drawing (A), a DNA molecule near the end of its replication is shown, with a short stretch of the molecule remaining unreplicated. If unravelling of the parental strands in this unreplicated segment is incomplete before the strands are completely unpaired and copied, then the residual intertwists between the parental strands would be converted to intertwines between the newly replicated progeny molecules (B and C) (Sundin & Varshavsky, , ; Varshavsky et al. ). These interwoven DNA duplexes must be resolved if the progeny molecules are to be segregated into two newly divided cells. Historically, replication had inspired much of the earlier considerations on the topological problems of DNA. It is now well-known that the necessity of disentangling DNA strands or duplexes also arises in nearly all other cellular transactions of DNA, including transcription, chromosome condensation and decondensation, and recombination. These aspects have been reviewed elsewhere (see Wang, , and references therein). As illustrated by the two examples summarized above, many of the topological problems of DNA are deeply rooted in its double-helix structure.
. Entering the DNA topoisomerases Nature invented a family of enzymes to solve the topological problems of DNA. These enzymes, termed the DNA topoisomerases (Wang & Liu, ), catalyse the interpenetration of DNA strands or double helices. In their presence, DNA strands and double helices can go through one another as if there were no physical boundaries in between. Since the discovery of the first member of this family of enzymes in the bacterium Escherichia coli nearly three decades ago (Wang, ), all organisms have been found to possess several of these enzymes. The limited scope of this review does not permit even a cursory coverage of the topoisomerase literature; references on earlier studies can be found in a number of monographs (Cozzarelli & Wang, ; Bates & Maxwell, ; Liu, a, b). The DNA topoisomerases perform their magic through transient breakage of DNA strands. They can be divided into two types: the type I enzymes break one DNA strand at a time and the type II enzymes both strands of a double helix in concert (Brown & Cozzarelli, ; Liu et al. ). The type I enzymes can be further divided into two subfamilies: the IA subfamily represented by bacterial DNA topoisomerases I and III and eukaryotic DNA topoisomerase III, and the type IB subfamily by eukaryotic DNA topoisomerase I and pox virus DNA topoisomerases. Members of the same subfamily are closely related in their amino acid sequences and reaction characteristics, and members of different subfamilies A simple molecular machine share little sequence homology and are mechanistically distinct (for reviews, see Wang, , and references therein). The type II DNA topoisomerases are thought to form a single subfamily, but recent studies of an enzyme DNA topoisomerase VI from archaeal hyperthermophiles suggest that the type II enzymes may also be divided into two subfamilies IIA and IIB (Bergerat et al. ): subfamily IIB is represented by archaeal DNA topoisomerase VI, and subfamily IIA by all other type II topoisomerases, including bacterial gyrase (DNA topoisomerase II), bacterial DNA topoisomerase IV, yeast and Drosophila DNA topoisomerase II, mammalian DNA topoisomerases IIα and IIβ, and T- even phage DNA topoisomerases. Because mechanistic analysis of archaeal DNA topoisomerase VI is still in its infancy, this fascinating enzyme will not be discussed in this review. It is plausible, however, that the archaeal enzyme may share many of the mechanistic features to be described and discussed below. All DNA topoisomerases catalyse transient DNA strand breakage by transesterification, a mechanism first postulated in (Wang, ). For the type I enzymes, the phenolic oxygen of an active-site tyrosyl residue undergoes transesterification with a phosphoryl group in a DNA strand, breaking the DNA phosphodiester bond and forming a phosphotyrosine linkage (Tse et al. ; Champoux, ). Rejoining of the DNA strand occurs through an apparent reversal of the DNA breakage reaction. Because the enzyme–DNA complex is likely to undergo large conformation changes between the DNA breakage and rejoining steps (Lima et al. ), the two steps are not necessarily the exact microscopic reversal of each other (Stivers et al. ). For DNA topoisomerases other than the type IB enzyme, the tyrosyl residue becomes linked to a DNA h- phosphoryl group in the formation of the covalent intermediate (Fig. ); for the type IB enzymes, it becomes linked to a h-phosphoryl group. The type I enzymes act as monomers in their breakage and rejoining of DNA strands one at a time; the type II enzymes are dimeric, and a pair of active-site tyrosyl residues undergo transesterification with a pair of phosphoryl groups in the two strands of a duplex DNA (Morrison and Cozzarelli, ; Sander & Hsieh, ; Liu et al. ).
. Type II DNA topoisomerases In this review, the focus is on the reaction mechanism of the type II DNA topoisomerases. The type II enzymes are of special interest in a number of ways. They unlink DNA catenanes and resolve intertwined chromosome pairs during mitosis, and in their absence cells die (reviewed in Yanagida & Sternglanz, ). These enzymes have been identified as the targets of a large number of natural toxins, antimicrobial agents, and anti-tumour therapeutics (Liu, b; Maxwell, ). They are also DNA-dependent ATPases, and how they couple their manipulation of DNA to ATP binding and hydrolysis raises fascinating mechanistic questions. The first members of the type II subfamily of DNA topoisomerases, bacterial DNA gyrase (DNA topoisomerase II), was discovered by Gellert et al.()as J. C. Wang
5′ DNA
O O PDNAO CH2 3′ O
′ OH 5 DNA
3′ OH O CH2 O P O CH2 DNA 3′
O
CH2
Fig. . Formation of a covalent intermediate between a DNA and a DNA topoisomerase. Nucleophilic attack of an enzyme tyrosyl group on a DNA backbone phosphorus leads to the breakage of the DNA strand and the simultaneous formation of a phosphotyrosine bond between the enzyme and the DNA. In the illustration shown, the phenolic oxygen of the tyrosyl group is attacking from the opposite side of a h-oxygen, and the enzyme tyrosyl becomes linked to a DNA h-phosphoryl group in the covalent intermediate. Such a covalent intermediate has been identified in reactions catalyzed by the type IA and type II DNA topoisomerases; for the type IB DNA topoisomerases, the enzyme tyrosyl group attacks from the opposite site of a h-oxygen and becomes linked to a DNA h-phosphoryl group in the covalent intermediate. an ATP-dependent DNA negative supercoiling activity. The term negative supercoiling refers to reducing the linking number between the two strands of a duplex DNA ring. If a DNA ring is in its most stable structure (termed a relaxed DNA), its linking number Lkm at a temperature of – mC and in a dilute aqueous buffer around neutral pH is readily estimated to be N\n, where N is the size of the DNA ring in base pairs, and n is the helical periodicity of a typical DNA under these conditions (Wang, ; Rhodes & Klug, ). If Lk of a DNA ring deviates significantly from Lkm, torsional and flexural strains are introduced into the molecule, and the DNA ring becomes contorted in space much like the deformation of a torsionally unbalanced rope. It is this contorted A simple molecular machine
(A) (B) (C) 1 409 679 1201 1428 ATP Y* (dimer)
Yeast DNA topoisomerase II
1804 1 875
GyrB GyrA
39 60 52
Phage T4
Fig. . A schematic alignment of the amino acid sequences of three representative type II DNA topoisomerases. Yeast DNA topoisomerase II is composed of a single subunit, E. coli DNA gyrase of two (GyrA and GyrB), and phage T DNA topoisomerase of three (encoded by genes , , and ). A, B, and C along the yeast polypeptide denote three protease- sensitive sites. For the yeast enzyme, the region between sites A and B, corresponding to the C-terminal half of the E. coli GyrB protein, is termed the Bh subfragment; the region between B and C, corresponding to the N-terminal two thirds of the E. coli GyrA protein, is termed the Ah subfragment. Thick lines represent regions with significant homology and thin lines regions with marginal homology. The approximate locations of the ATPase site, the active-site tyrosine, and residues that form the primary dimer interface in the yeast enzyme are indicated by ATP, Y*, and (dimer) above the line representing the enzyme. Illustration drawn after fig. in Berger & Wang (). shape that inspired the term supercoiling, supertwisting, or superhelix formation. By definition, a negatively supercoiled DNA is one with Lk ! Lkm, and a positively supercoiled DNA one with Lk " Lkm. DNA gyrase can apparently utilize the chemical energy of ATP hydrolysis to drive a DNA ring to an energetically unfavourable state with an Lk ! Lkm. The discovery of bacterial DNA gyrase was soon followed by the discovery of phage T DNA topoisomerase (Liu et al. ; Stetler et al. ) and eukaryotic DNA topoisomerase II (Baldi et al. ; Hsieh & Brutlag, ; Miller et al. ). The phage and eukaryotic enzymes differ from bacterial DNA gyrase in that they catalyse the relaxation of a positively or negatively supercoiled DNA in the presence of ATP, but not DNA supercoiling. These enzymes also differ in their quaternary structures. Bacterial gyrases are comprised of two subunits (Gellert et al. ), the eukaryotic enzymes one (Miller et al. ; Sander & Hsieh, ; Goto & Wang, ), and the phage T enzyme three (Liu et al. ; Stetler et al. ). The amino acid sequences of the enzymes showed clearly, however, that the enzymes are closely related (see for example, the compilation of Caron & Wang, ); a schematic alignment of the amino acid sequences of three type II DNA topoisomerases of different quaternary structures is illustrated in Fig. . J. C. Wang
. . Transporting one DNA double helix through another A breakthrough in understanding the mechanism of the type II DNA topoisomerases came around . In earlier years, it was assumed that in reactions catalysed by all topoisomerases only one of the two strands of a DNA double helix would be transiently broken so that the intact strand could hold the broken ends close to each other for their subsequent rejoining. Several lines of evidence showed, however, that bacterial DNA gyrase and phage T DNA topoisomerase do not act in this fashion. First, it was observed in that the addition of a protein denaturant to a plasmid-bound gyrase would linearize rather than nick the plasmid in the presence of nalidixate, an antibiotic that targets bacterial gyrase (Sugino et al. ; Gellert et al. ). Furthermore, a protein moiety was found to be covalently attached to each of the two h ends of the linear DNA produced in this reaction (Morrison & Cozzarelli, ), and this protein moiety was shown to be the A-subunit of gyrase (Tse et al. ; Sugino et al. ). These findings demonstrated that the double-stranded breakage of DNA by gyrase is a consequence of covalent adduct formation between the enzyme and both strands of a duplex DNA. Second, it was found that the bacterial and phage enzymes can interconvert several novel topological forms of duplex DNA rings, for examples the unlinking of DNA catenanes or the knotting of unknotted rings (Liu et al. ; Kreuzer & Cozzarelli, ; Mizuuchi et al. ). These topological transformations strongly suggested that the bacterial and phage enzymes can catalyse the passage of one DNA double helix through a transient double-stranded break in another. Third, quantitative measurements of linking number changes of DNA rings by DNA gyrase or phage T DNA topoisomerase showed that these enzymes alter Lk in units of two (Brown & Cozzarelli, ; Liu et al. ; Mizuuchi et al. ). Such an even-number change in Lk can best be interpreted in terms of a mechanism in which an enzyme mediates the passage of one DNA segment through another of the same DNA ring during each round of reaction (Brown & Cozzarelli, ; Liu et al. ; Mizuuchi et al. ). It turned out that the dyadic change in Lk by such an operation was foretold in a mathematical analysis of the linking number between the two edges of a closed ribbon (Fuller, ), and could also be inferred from earlier examples illustrating the topological properties of a DNA ring (Crick, ). Studies of eukaryotic type II DNA topoisomerases also led to the same conclusion that these enzymes act by a double-stranded DNA breakage, passage, and rejoining mechanism (Baldi et al. ; Hsieh & Brutlag, ). Once it was recognized that the type II enzymes transiently cleave double- stranded DNA, it was straightforward to determine the relative positions of the pair of cleavage sites such an enzyme would make in a DNA duplex. From these measurements, DNA breakage by a type II topoisomerase was shown to involve transesterification between the active-site tyrosyl residues of the enzyme and a pair of staggered phosphoryl groups four base pairs apart (Morrison & Cozzarelli, A simple molecular machine ; Sander & Hsieh, ; Liu et al. ). In the case of bacterial DNA gyrase, the A-subunit is often referred to as the ‘DNA breakage-and-rejoining subunit’ because of the presence of an active-site tyrosyl group in each A-subunit (Tse et al. ; Sugino et al. ; Horowitz & Wang, ). However, essential residues from both A- and B-subunits are most likely present in each catalytic pocket for transesterification. Whereas purified DNA gyrase A-subunit has no detectable DNA cleavage\rejoining activity (Higgins et al. ), the combination of the A-subunit and a C-terminal fragment of the B-subunit does (Brown et al. ; Gellert et al. ). Similarly, yeast DNA topoisomerse II lacking the ‘GyrB’ portion of the polypeptide has no DNA cleavage activity, but a fragment comprised of amino acid residues to about , corresponding to the C- terminal half of the gyrase B-subunit plus the N-terminal two thirds of the gyrase A-subunit, is capable of forming enzyme–DNA covalent adduct (Berger et al. ). Site-directed mutagenesis studies of yeast DNA topoisomerase II further confirmed the requirement of residues in both Ah and Bh domains of the enzyme (Q. Liu and J. Wang, to be published; see the legend to Fig. for the boundaries of the Ah and Bh domains).
. The clamp model: a type II DNA topoisomerase as an ATP-modulated protein clamp From the above description, a type II DNA topoisomerase presumably binds to a double-stranded DNA segment, to be termed the gate-segment or G-segment, with the pair of active-site tyrosyl residues of the enzyme positioned near a pair of DNA phosphorus atoms for creating an opening or gate in the DNA double helix. The DNA-bound enzyme must then capture a second double-stranded DNA segment, termed the transported-segment or T-segment, in order to move it through the G-segment. How does this occur? Studies carried out between the mid s and early s led to the proposal of the clamp model, in which a type II DNA topoisomerase is assumed to act as an ATP-modulated protein clamp (Roca & Wang, ). According to this model, the enzyme possesses a pair of jaws, which come into contact to close a molecular gate upon binding of ATP, and come apart to re-open the gate upon ATP hydrolysis and product release. The closing and opening of the jaws can occur either in a free enzyme, or in one bound to a G-segment. The ATP-modulated cycling time is faster when the enzyme is DNA-bound, and the magnitude of this increase in rate can be deduced from the DNA dependence of the rate of ATP hydrolysis. In the case of yeast DNA topoisomerase II, the Michaelis constant KM −" and the apparent turnover number kcat were measured to be n m and n s per dimeric enzyme in the absence of DNA (at mC in a medium containing m Tris acetate, pH n, m potassium acetate, m magnesium acetate, and m-mercaptoethanol). In the presence of DNA, positive cooperativity between the two ATPase sites was observed. The concentration of ATP at half- maximal velocity drops to about n m, and kcat is increased by about -fold (Lindsley & Wang, a). Thus at saturating ATP concentrations the motion of J. C. Wang
G G
+ATP T
Fig. . The protein clamp model of a type II DNA topoisomerase. An enzyme molecule is depicted as a dimeric protein clamp bound to a DNA segment termed the gate or G- segment. According to this model, closing and opening of the pair of jaws of the protein clamp are modulated by ATP binding and hydrolysis and release of the hydrolytic products. A second DNA segment, termed the transported or T-segment, can enter the DNA-bound protein clamp; ATP-mediated closure of the jaws captures the T-segment and drives it through the transiently opened DNA gate in the G-segment. Drawing taken from Roca & Wang ().
clamp opening and closing is probably accelerated by about -fold when the yeast enzyme is bound to DNA. For E. coli DNA gyrase, binding to DNA was also found to stimulate the ATPase activity of the enzyme (Sugino et al. ; Sugino & Cozzarelli, ; Staudenbauer & Orr, ; Maxwell & Gellert, ). The ATP-modulated opening and closing of the enzyme clamp is assumed to be directly related to the type II topoisomerase-mediated transport of one DNA double helix through another. When a DNA-bound protein clamp is in the open state, a T-segment can enter through the open jaws into the enzyme interior. This entrance is presumably facilitated by weak DNA–protein interactions and is also influenced by the topology of the DNA substrate. Closing of the jaws by ATP-binding then captures the T-segment and drives it through the G-segment (Fig. ). The clamp model was first hinted by images of DNA gyrase molecules in electron micrographs (Kirschhausen et al. ). Experimental evidence supporting the model came initially from studies of DNA binding by yeast DNA topoisomerase II in the absence and presence of h-adenylyl-β,γ- imidodiphosphate (ADPNP), a non-hydrolysable β,γ-imido analogue of ATP. In the absence of the nucleotide, the yeast enzyme binds readily to linear or circular DNA, with a slight preference for supercoiled DNA (by about a factor of ). Pre- incubation of the enzyme with ADPNP, however, converts it to a form capable of binding linear DNA but not any form of DNA rings (Roca & Wang, ). The specific binding of linear DNA to the ADPNP–enzyme complex is not caused by interactions between the DNA ends and the enzyme, as the ends of an enzyme- bound linear DNA can be readily joined by DNA ligase. Thus it appeared that the A simple molecular machine binding of ADPNP to the enzyme converts it to an annular form with a hole sufficiently large for a linear DNA to thread through (Roca & Wang, ). Results of similar experiments with E. coli DNA gyrase were, however, not as clear cut (A. Maxwell and M. Gellert, personal communications); it is plausible that measurements with the bacterial enzyme are subject to complications arising from the association\dissociation of the DNA gyrase subunits. In a later section, mechanistic differences between DNA gyrase and other type II DNA topoisomerases will be discussed. The clamp model explains readily an earlier observation with Drosophila DNA topoisomerase II that addition of ADPNP makes the Drosophila enzyme resistant to dissociation by salt if it is bound to a DNA ring, but not if it is bound to a linear DNA (Osheroff, ). For a type II topoisomerase bound to a DNA ring, closing the protein clamp by ADPNP would introduce a salt-resistant topological link between the protein and DNA (Pommier et al. ; Roca & Wang, ). The same interpretation explains why a linear DNA bound to the enzyme–ADPNP complex is readily dissociated from the complex upon exposure to high salt, but ligation of the ends of the linear DNA before salt addition prevents its separation from the protein (Roca & Wang, ). According to the clamp model, the jaws of a type II DNA topoisomerase bound to a DNA G-segment must close after the entrance of the T-segment in order to effect the transport of the T-segment through the G-segment. This notion was supported by an order-of-mixing experiment in which two different DNA rings and ADPNP were added sequentially to yeast DNA topoisomerase II. Under conditions such that an enzyme-bound DNA could not be displaced by a subsequently added second DNA, it was shown that the G-segment always resides in the first DNA ring added, and catenation between the two sequentially added DNA rings is possible only if the second ring is added before the addition of ADPNP (Roca & Wang, ).
. The number of gates in the protein clamp A protein clamp has at least one molecular gate which closes when the jaws of the clamp come into contact and opens when they come apart. The question was raised, however, about whether there are separate gates for the entrance and exit of the T-segment, or there is only a single gate through which the T-segment would first enter and later exit (Roca & Wang, ). Fig. illustrates the two situations. In each case, an ATP-modulated enzyme clamp bound to a G-segment is shown, and a T-segment can enter the clamp in its open state as described in the section above. In the two-gate model (a), closing the jaws denoted by N drives the T-segment through the transiently opened G-segment and out of a second protein gate on the opposite side of the entrance gate [right-side drawing in (a)]. In the one-gate model (b), closing of the protein clamp drives the T-segment through the G-segment into a holding dock; following ATP-hydrolysis, the clamp re-opens for the exit of the T-segment through the same gate it had entered earlier. As illustrated in the right-side drawing in (b), in the one-gate model at least a part of J. C. Wang
(a) T
G G
T
NN NN
T
(b)
T
G
NN
T
Fig. . The two-gate (a) and one-gate (b) model of DNA transport by a type II DNA topoisomerase. See the text for explanation. Illustration taken from Roca & Wang (). the G-segment must dissociate from the enzyme prior to the exit of the T- segment, because backtracking of the T-segment through the G-segment would mean no net transport. In the two-gate model, exist of the T-segment requires transient disruption of protein–protein interaction between the pair of jaws constituting the exit gate; in the one-gate model, transient disruption of DNA-protein interaction is involved instead. The two-gate model was first proposed in for DNA gyrase (Mizuuchi et al. ; Wang et al. ). Because gyrase interacts with about bp of the DNA G-segment (Liu & Wang, a, b; Klevan & Wang, ; Kirkegaard & Wang, ; Morrison & Cozzarelli, ; Fisher et al. ; Rau et al. ; Orphanides & Maxwell, ), in the absence of experimental data it appeared that separating two interacting protein domains would be no more difficult than dissociating a long stretch of DNA from the protein surface. The first attempt to distinguish the two models was made more recently (Roca & Wang, ). A series of experiments were carried out to examine the unlinking of two singly linked DNA rings by yeast DNA topoisomerase II upon A simple molecular machine
G ATP T 2 NN 3
T T
1 G G
NN NN NN
T 4 5 G ADP+Pi
NN
Fig. . Reaction steps in the two-gate model of ATP-dependent DNA transport by a type II DNA topoisomerase. An enzyme molecule in its open-clamp conformation can bind a G- segment, and the jaws labelled N in the drawings can be in the open or closed state depending on the absence or presence of enzyme-bound ATP. A T-segment can enter the enzyme when it is in the open-clamp conformation. The closure of the enzyme clamp upon ATP-binding traps the T-segment and forces it to first go through the DNA gate and then an exit gate on the opposite side of the entrance gate (the N-gate) formed by the pair of jaws labelled N. the addition of ADPNP. Because the non-hydrolysable nucleotide can trigger the closure of the N-gate but does not allow its re-opening, the one-gate model would predict that both of the unlinked DNA rings would be topologically linked with the annular enzyme. The two-gate model, on the other hand, would predict that only the DNA ring containing the G-segment would be bound in such fashion, as the ring containing the T-segment would be expelled from the enzyme through the exit gate. The results of the experiments are in agreement with the two-gate model: decatenation of the singly linked rings was found to occur readily upon the addition of ADPNP, and analysis of the products showed that the ring in which the T-segment resided was free in solution and the ring in which the G-segment resided remained topologically linked to the annular enzyme (Roca & Wang, ). Incorporation of this finding into the ATP-modulated protein clamp model of type II DNA topoisomerases led to the model depicted in Fig. .
. Three-dimensional structures of type II DNA topoisomerase fragments The structures of several type II DNA topoisomerase fragments have been solved by X-ray crystallography in recent years (Wigley et al. ; Berger et al. ; Lewis et al. ; Morais Cabral et al. ). Fig. depicts a n AH structure of J. C. Wang a -kDa N-terminal fragment of the B-subunit of E. coli DNA gyrase (Wigley et al. ). This fragment, comprised of amino acid residues –, contains the ATPase domain of the enzyme. The crystal was obtained in the presence of ADPNP, and there is one bond ADPNP molecule in each half of the dimeric protein molecule; therefore the structure is likely to resemble the ATP-bound form of the protein fragment. Two lysyl side-chains lining the nucleotide-binding pocket, Lys- and Lys-, were previously identified to form covalent links with an ATP affinity analogue pyridoxol h-diphospho-h-adenosine (Tamura & Gellert, ). Several studies showed that the -kDa E. coli GyrB fragment is monomeric in solution but dimerizes upon binding of ADPNP, but not ADP (Ali et al. , ). Measurements of ATP hydrolysis by E. coli DNA gyrase (Maxwell et al. ) and yeast DNA topoisomerase II (Lindsley & Wang, a), as well as measurements of ADPNP-binding by DNA gyrase (Tamura et al. ), also suggest a cooperative interaction between the two nucleotide binding sites. Taken together, these results indicate that the binding of an ATP to one of the ATPase catalytic pockets of a type II DNA topoisomerase triggers a conformation change of the ATPase domain, which in turn leads to intramolecular dimerization of the domains and facilitates the binding of a second ATP. An important conclusion that can be drawn from the crystallographic and biochemical experiments is that ATP hydrolysis can not occur without dimerization of the ATPase domains of the enzyme (Wigley et al. ; Tamura et al. ; Ali et al. ). This point will be elaborated upon in a later section on the coupling between ATP usage and DNA transport by the type II DNA topoisomerases. Because ADPNP binding is known to trigger the closure of the enzyme clamp in the case of yeast DNA topoisomerase II, from the above discussion it can be deduced that the pair of ATPase domains in a type II DNA topoisomerase constitute the entrance gate; this gate has been termed the N-gate (Figs and ) because in a single-polypeptide eukaryotic enzyme the ATPase domain is close to the N-terminus. In the structure shown in Fig. (Wigley et al. ), an N-terminal arm (amino acid residues –) of the DNA gyrase B-subunit extends from the ATPase domain surface to form dimer contacts with the other monomer. The C-terminus proximal helix (residues –) also extends from the core of the C-terminal subdomain of the structure to contact the symmetry-related helix of the other monomer. This C-terminal interface is likely to represent a crystallographic rather than native protein contact, however. Fig. depicts a n AH crystal structure of a -kDa fragment of yeast DNA topoisomerase II spanning residues – (Berger et al. ). This fragment lacks the ATPase subfragment as well as amino acid residues at the C- terminus of the intact enzyme, but it is capable of covalent adduct formation with double-stranded DNA. From the alignment shown in Fig. , the – fragment can be subdivided into two parts, termed the Bh subfragment (residues to about ), which corresponds to the C-terminal half of the gyrase B- subunit, and the Ah subfragment, which corresponds to the gyrase A-subunit A simple molecular machine lacking the -residue C-terminal fragment. In the cases of E. coli DNA gyrase and yeast and Drosophila DNA topoisomerase II, this missing C-terminal segment is known to be dispensable for catalysis of DNA transport (Reece & Maxwell, a; Shiozaki & Yanagida, ; Crenshaw & Hsieh, ; Caron et al. ; Kampranis & Maxwell, ). The dispensability of the C-terminal segment is also consistent with its absence in phage T and T DNA topoisomerase (see Fig. ). Therefore, in combination, the structures of the gyrase ATPase subfragment and the yeast DNA topoisomerase II BhAh fragment provide a D sketch of a functional type II DNA topoisomerase. In the heart-shaped (BhAh)# structure of the yeast enzyme fragment, the two Bh subfragments contact each other to form an arch, which caps the V-shaped Ah–Ah dimer to enclose a large hole AH wide at its base. The two Ah polypeptides contact at their C-terminal region to form the Ah–Ah dimer interface. This interface represents the major contact between the two BhAh halves and it buries # over AH of surface. Three of the four known yeast top mutations that result in cold-sensitivity of the mutant cells were mapped to this interface (Thomas et al. ). The Bh–Bh contacts are less extensive but appear significant (Berger et al. ). A pair of semicircular grooves formed by residues from both the Ah and Bh subfragments are present near the top of the V-shaped Ah–Ah dimer. These grooves, with an approximately AH opening, are decorated with positive surface charges and have been implicated in the binding of the G-segment (Berger et al. ). A double-stranded DNA segment with a -nucleotide h-overhang can be modelled into each groove in the protein structure, with the single-stranded nucleotides extending through a narrow tunnel and joining to the active-site tyrosyl residue Tyr- (Fig. ). Thus the -kDa structure is believed to correspond closely to the conformation of the enzyme after it has cleaved the G- segment and pulled it apart (Berger et al. ). In the proposed model, a part of the DNA-binding surface is formed by a domain comprised of residues –. This domain has the same fold as the DNA binding domain of the E. coli catabolite activator protein (CAP) and histone H, and has been termed the CAP domain (Berger et al. ). Recent lysine footprinting data are consistent with the assignment of the G-segment binding surface described above (Li & Wang, ). Fig. illustrates a n AH structure of a -kDa fragment (residues –)ofE. coli DNA GyrA protein (Morais Cabral et al. ). As expected from amino acid sequence comparisons (Fig. ), the overall architecture of this fragment is very similar to that of the Ah subfragment of yeast DNA topoisomerase II (Fig. ). There are significant differences between the relative positions of the various domains within the E. coli and the yeast enzyme fragment, however. Whereas the pair of CAP domains in the yeast enzyme are well-separated, they are rotated from their positions in the yeast enzyme and brought into contact to form the ‘head dimer interface’ in the gyrase structure shown in Fig. (Morais Cabral et al. ). The three long helices (α, α, and α) connecting the N-proximal core and the primary dimer interface also adopt different conformations in the yeast and E. coli fragment structures, which will be discussed in a later section. J. C. Wang
. How are the various subfragments connected? No high-resolution structure of a type II DNA topoisomerase capable of moving one DNA through another is presently available. Electron microscopic examination of E. coli gyrase and its subunits (Kirschhausen et al. ), human DNA topoisomerase II (Schultz et al. ), and intact and fragments of yeast DNA topoisomerase II (Benedetti et al. ) has provided low-resolution information on the organization of the subdomains. Visualization of negatively stained human enzyme by scanning transmission electron microscopy, for example, showed a V-shaped tripartite structure with a dense globular domain, about AH in diameter, flanked by two smaller spheres about – AH in diameter (Schultz et al. ). The pair of smaller spheres at the opening of the V-shaped molecule appeared to be connected to the larger globular body by linkers – AH in length that were barely visible in the micrographs, and the angle extended by the smaller spheres was typically in the range of –m. A similar tripartite structure was seen by transmission microscopy with metal-shadowed yeast DNA topoisomerase II (Benedetti et al. ). Deletion of the N-terminal residues of the yeast enzyme resulted in a large decrease in the average size of the smaller spheres in the tripartite structures (Benedetti et al. ), indicating that these spheres represent the N-terminal or the GyrB-halves of the dimeric enzyme. Interestingly, with either the human or the yeast enzyme, preincubation of the enzyme with ADPNP caused a major change in the appearance of the images; in the majority of molecules, the two smaller spheres appeared to be in contact with each other following incubation with the ATP analogue (Schultz et al. ; Benedetti et al. ). Micrographs of the human enzyme showed a preponderance of bipartite structures in the presence of ADPNP, with a larger globular structure of about AH in diameter and a smaller one of about AH in diameter (Schultz et al. ). In combination, the X-ray crystallography and electron microscopy results suggest that each polypeptide of eukaryotic DNA topoisomerase II is organized into two modules A and B, corresponding approximately to the A- and B-subunits of gyrase. The pair of A modules in a dimeric enzyme are normally in contact with each other in the presence or absence of bound ATP. The B modules, on the other hand, form strong contacts only upon binding of ADPNP (and by implication ATP); in the absence of bound ATP, the pair of B modules often stay apart. Remarkably, there seem to be few protein–protein contacts between the A and B modules in the absence of DNA, as suggested by the separation of the two modules in electron micrographs of the molecules (Schultz et al. ; Benedetti et al. ), and by the results of protein footprinting experiments designed to test whether any lysyl residue in the GyrA or GyrB half of yeast DNA topoisomerase II might be protected against citraconylation by the presence of the other half of the polypeptide (Li & Wang, ). The structural studies, especially the crystal structures of the -kDa yeast DNA topoisomerase II fragment and the -kDa E. coli DNA gyrase A subunit fragment, also reveal a high degree of flexibility in the detailed positioning of the A simple molecular machine various domains of the type II DNA toposiomerases. This structural variability is presumably related to the ability of a type II DNA topoisomerase to assume very different conformations during its catalytic steps. At the same time, this structural flexibility increases the difficulty of reconstructing the various conformational states of a functional enzyme from the structures of its parts.
. A molecular model of DNA transport by a type II DNA topoisomerase The structural data summarized above provided additional support of the model depicted in Fig. and added much molecular details to the model. Fig. illustrates a refined model combining the known biochemical and structural data (Berger et al. ). The particular enzyme in this illustration is that of yeast DNA topoisomerase II lacking the C-terminal amino acid residues that are dispensable for its catalytic actions. It is most likely, however, that the basic features of the model are shared by all type II DNA topoisomerases. In a later section, the plausible difference between bacterial DNA gyrase and type II enzymes exemplified by yeast DNA topoisomerase II will be discussed. The structure denoted by in Fig. is a representation of the free enzyme in an open-clamp conformation. The purple (Ah)# structure in is the same as that seen in the -kDa crystal structure except that the pair of Bh subfragments with the ATPase subfragments tagged on, are not in contact. Such an open conformation with detached Bh subfragments is presumably one of the major conformations of the free enzyme, and this open conformation is necessary for the binding of the DNA G-segment (shown as a rod above the enzyme). Upon binding of the DNA G-segment, there is a significant change in the conformation of the enzyme (). The two enzyme halves move toward each other by a large distance, estimated to be – AH (Berger et al. ), and the pair of active-site tyrosyl residues are now positioned near the scissile phosphates for nucleophilic attack. The structure of the purple (Ah)# dimer depicted in resembles more the one shown in Fig. for the Ah dimer of E. coli gyrase (Morais Cabral et al. ) than the one shown in Fig. for the -kDa fragment of the yeast enzyme. The precise positions of the Bh subfragments in are uncertain. It has been suggested (Berger et al., ) that these subfragments may be oriented very differently from their positions in the -kDa fragment structure of Berger et al.(). The pair of ATPase subfragments in can combine or separate depending on the state of ATP binding, and a T-segment can enter the enzyme clamp when these jaws are apart. Closure of the jaws upon the binding of ATP would then trap the T-segment and initiate a conformational cascade in the enzyme-DNA complex: the DNA G-segment is transiently broken and splayed apart, and the T-segment is forced through the DNA gate into the large cavity below (illustrations to in Fig. ). The conformation of the (BhAh)# part of the enzyme shown in of Fig. is that seen in the crystal structure of the -kDa fragment of the yeast enzyme. When the two enzyme halves retract toward each other following the passage of the T- segment through the DNA gate, the size of the cavity containing the T-segment J. C. Wang is reduced and the T-segment exits the enzyme through the Ah–Ah dimer interface (illustration of Fig. ). The Ah–Ah interface has been termed the C-gate because its constituents are close to the carboxyl termini of the Ah polypeptides. The open state of the C-gate is probably a transient one. Following the exit of the T-segment and closure of the C-gate, the N-gate can re-open after ATP hydrolysis and the release of the hydrolytic products ( in Fig. ), and the enzyme is posed for another cycle of DNA transport. The availability of detailed structural information had also made it possible to test more directly a key postulate of the two-gate protein clamp model, namely the role of the C-gate for the exit of the T-segment (Roca et al. ). Based on the crystal structure of the -kDa fragment of the yeast enzyme, site-directed mutagenesis was carried out to replace Lys- and Asn- by cysteines. In the mutant enzyme, a pair of disulphide bonds can form across the Ah–Ah dimer interface to lock the C-gate. When the single-step decatenation experiment of Roca & Wang () was repeated with the mutant enzyme, with its C-gate locked by the formation of disulphide bonds across the Ah–Ah interface, the unlinked DNA rings following one round of DNA transport were found to remain topologically linked to the enzyme–ADPNP complex (Roca et al. ). Furthermore, exit of the DNA ring containing the T-segment was observed upon subsequent addition of -mercaptoethanol, which reduces the disulphide bonds and hence unlocks the C-gate, to the reaction products (Roca et al. ). These and other data, while providing strong evidence in support of the two-gate mechanism, do not preclude the possibility that under certain conditions (for example when the G-segment can dissociate readily from the enzyme) a type II DNA topoisomerase could promote DNA transport by a one-gate mechanism (Lindsley, ). A special one-gate mechanism to account for the ATP- independent relaxation of negative supercoiled DNAs by bacterial DNA gyrase will be discussed in Section ...
. A type II DNA topoisomerase can be viewed as a molecular machine that utilizes ATP as the fuel in the transport of one DNA double helix through another. From a thermodynamic point of view, only DNA supercoiling by bacterial DNA gyrase, and not the removal of supercoils by the other type II DNA topoisomerases, is expected to require a high-energy cofactor like ATP. It is clear, however, that kinetically the transport of DNA by a type II DNA topoisomerase is strongly dependent on ATP. Removal of DNA supercoils by yeast DNA topoisomerase II is undetectable in the absence of ATP, and mutating a critical ATPase domain residue Gly- of the enzyme to alanine also abolishes its DNA relaxation activity in the presence of ATP (Linsley & Wang, a). A low level of DNA transport activity in the absence of ATP is present, however, in several type II DNA topoisomerases including E. coli DNA gyrase (Gellert et al. , ; Sugino et al. ; Higgins et al. ; Kreuzer & A simple molecular machine Cozzarelli, ; Mizuuchi et al. ; Krasnow & Cozzarelli, ; Marians, ), phage T DNA topoisomerase (Liu et al. , ) and Drosophila DNA topoisomerase II (Osheroff et al., ). A derivative of gyrase lacking the ATPase domain was also found to relax supercoiled DNA in the presence or absence of ATP (Brown et al. ; Gellert et al. ). In the sections below, the available information on how a type II DNA topoisomerase couples ATP usage to DNA transport is summarized. Attempts are made, at the risk of oversimplification, to interpret this intricate coupling in terms of the known or predicted structural features of the various enzyme–DNA complexes. The low level ATP-independent DNA transport activity of DNA gyrase will be discussed in a separate section.
. ATP utilization Because the binding of the non-hydrolysable ATP analogue ADPNP to a type II topoisomerase can effect one round of DNA transport, it is generally assumed that DNA transport by the enzyme is driven by ATP binding, and ATP hydrolysis and product release are only necessary for enzyme turnover (Sugino et al. ; Peebles et al. ). It is plausible, however, that ATP hydrolysis may further accelerate the coupled process. An example is provided by the recent study of the roles of nucleotide cofactors in the reaction steps catalysed by the elongation factor G, a GTPase involved in the transloction of bacterial ribosomes along messenger RNA (Rodnina et al. ). It is known that non-hydrolysable GTP analogues can effect one round of translocation. With GTP itself, however, rapid hydrolysis of the nucleotide was found to precede translocation. These observations suggest that although the binding of a non-hydrolysable GTP analogue can effect translocation, GTP hydrolysis may further accelerate the rearrangements of the ribosome that drive translocation (Rodnina et al. ). The intricacy of ATP utilization in the transport of DNA by the type II DNA topoisomerases is illustrated by the recent pre-steady state kinetic analysis of ATP hydrolysis by yeast DNA topoisomerase II (Harkins & Lindsley, a, b). As expected, the DNA-bound enzyme consumes two ATP per enzyme turnover event. These bound ATP molecules are hydrolysed sequentially, however. Rapid hydrolysis of one ATP and the release of the hydrolytic products appear to occur before the second ATP is hydrolysed. Deducing the structural changes accompanying this stepwise hydrolysis of bound ATP should provide further insight on the intricate coupling of ATP usage to DNA transport.
. How tight is the coupling between DNA transport and ATP binding and hydrolysis? .. Dependence of the coupling efficiency on the degree of supercoiling of the DNA substrate Measurements with yeast DNA topoisomerase II indicate that the coupling efficiency can be very high under optimal conditions. Quantitative analysis of linking number changes of DNA rings showed that for a moderately supercoiled J. C. Wang DNA, % of all bound enzyme molecules are able to promote one round of DNA transport upon the addition of excess ADPNP (Roca & Wang, ). Because a fraction of the bound enzyme molecules was probably not active, the actual coupling efficiency could be even higher and close to the theoretical limit of %. The same study showed that with a completely relaxed DNA ring the efficiency is considerably lower: about one out of four bound enzyme molecules would complete a DNA transport event upon ADPNP addition. Two sets of measurements of the coupling efficiency in the presence of ATP itself rather than its non-hydrolysable analogue were also carried out with the yeast enzyme (Lindsley & Wang, a). In one, parallel measurements of the rate of ATP hydrolysis and the rate of removal of DNA supercoils were carried out at a saturating ATP concentration of m. The reaction temperature was set below n mC so that the ATPase and DNA transport rates were slowed down sufficiently to permit manual quenching of the reactions. From these measurements it was calculated that about np ATP were hydrolysed per DNA transport event. When the same measurements were carried out at mC and lower ATP concentrations, however, the number of ATP hydrolysed per DNA transport event was measured to be as low as n, with an average of npn. These results suggest that at a high ATP concentration when ATP binding is rapid, closure of the enzyme clamp often occurs without trapping a T-segment; at a low ATP concentration, at least in the case of a supercoiled DNA the rates of ATP binding and the closure of the enzyme clamp are both slow relative to the rate of entrance of the T-segment into an enzyme in its open-clamp conformation, leading to a very high coupling efficiency. In terms of the number of ATP required for one round of DNA transport by a type II DNA topoisomerase, the theoretical limit is probably close to one ATP per DNA transport event at very low ATP concentrations: the binding of an ATP to one half of the dimeric enzyme could be sufficient in triggering dimerization of the N-terminal domain and the conformational cascade that follows, leading to the transport of a T-segment through the enzyme-bound G-segment. Studies with a heterodimeric yeast enzyme with one wild-type polypeptide and one mutant polypeptide with a mutation in the ATPase domain suggest that the binding of a single ATP to a dimeric enzyme is sufficient to trigger the closure of the enzyme clamp (Lindsley & Wang, b). At high ATP concentrations, however, the binding of ATP to wild-type yeast DNA topoisomerase II is cooperative, and the binding of an ATP to one protomer of the dimeric enzyme is likely to be followed rapidly by the closure of the N-gate and the binding of a second ATP to the other protomer (Lindsley & Wang, a). Therefore at high ATP concentrations the theoretical limit of the coupling efficiency is expected to be two ATP for each DNA transport event. For E. coli DNA gyrase, the apparent coupling efficiency was also found to be strongly dependent on DNA topology. With a positively supercoiled DNA, the coupling efficiency approaches %, and a linking number reduction of two per (BA)# molecule was observed upon the addition of ADPNP (Bates et al. ). The coupling efficiency decreases steadily with DNA substrates of increasing A simple molecular machine degrees of negative supercoiling, and the ADPNP-mediated reduction of Lk becomes undetectable for a moderately negatively supercoiled DNA with a specific linking difference of kn [Bates et al. ; the specific linking difference is defined as the fractional deviation of Lk from Lkm or (LkkLkm)\Lkm]. Earlier measurements also showed that the addition of the non-hydrolysable nucleotide to E. coli DNA gyrase bound to a relaxed DNA reduces Lk by about n per A protomer or n per dimeric enzyme molecule (Sugino et al. ), corresponding to an efficiency of about %. With ATP as the cofactor, earlier measurements of negative supercoiling of a relaxed DNA by gyrase suggested that the apparent coupling efficiency is high during the early phase of the reaction, but drops to zero when the specific linking difference of the DNA approaches kn (reviewed in Reece & Maxwell, b). The results summarised above show that in terms of the relaxation of a positively supercoiled DNA, the coupling efficiency for bacterial DNA gyrase is not very different from that for yeast DNA topoisomerase II. Because the yeast enzyme does not catalyse DNA negative supercoiling, it is not possible to compare the efficiencies of the two enzymes in this reaction. There are two complications in the interpretation of the coupling efficiencies of DNA negative supercoiling by gyrase. First, in nearly all measurements with the bacterial enzyme, only the net change in the average linking number of a DNA ring was examined. For a DNA gyrase bound to a negatively supercoiled DNA, the formation of a positive-noded configuration between the G-segment and the incoming T-segment is favoured by the DNA-enzyme binding energy, but disfavoured by the free energy of DNA supercoiling (see Section ..). Therefore the number of DNA transport events can be equated to one half of the net decrease in Lk only if the DNA–enzyme binding energy is the dominant term, otherwise ATP-triggered inversion of both positive and negative nodes may occur in a population of negatively supercoiled DNA molecules. Second, DNA gyrase is known to possess an ATP-independent DNA relaxation activity that is specific for negatively supercoiled DNA (Sugino et al. ; Gellert et al. , ; Higgins et al. ). This uncoupled pathway may significantly reduce the apparent coupling efficiency, especially if a highly negatively supercoiled DNA substrate is used. In experiments on the coupling efficiency of ADPNP triggered DNA transport in a positively supercoiled DNA, these complications are largely absent (the first complication can also be eliminated entirely by the use of a DNA substrate consisting of a single linking number isomer, so that the inversion of both positive and negative nodes can be measured directly from an analysis of the distribution of the linking numbers of the products; see Roca & Wang, ).
.. Coupling efficiency and DNA binding In terms of the two-gate protein clamp model, the above data can be understood by assuming that normally the binding of ADPNP or ATP to an enzyme bound to a G-segment always leads to the closure of the N-gate and the sequential transport of a T-segment, if one is present inside the enzyme, through the DNA gate and the protein exit gate. This interpretation is supported by two J. C. Wang observations. First, as shown by the single-step decatenation experiments of Roca & Wang (), the DNA ring containing the T-segment always exits the enzyme clamp following its passage through the DNA gate. Second, through the use of a mutant enzyme YF, which is incapable of opening the DNA gate owing to the replacement of the pair of active site tyrosyl residues by phenylalanines, it was found that the binding of ADPNP to a G-segment bound mutant enzyme can trigger the closure of the N-gate, but a second DNA ring can not be captured (J. Roca, W. Li, and J. Wang, unpublished). Together, these two observations demonstrate that the probability of capturing a T-segment without its being transported through the DNA gate and the exit gate of the enzyme is low. If the binding of ADPNP to a G-segment-bound enzyme is always followed by the transport of a T-segment that has entered the enzyme clamp, then the dependence of the overall coupling efficiency on the topology of the DNA substrate must reflect differences in the probability of finding a T-segment within an open enzyme clamp with DNA substrates supercoiled to different extents. For type II DNA topoisomerases resembling yeast DNA topoisomerase II, the coupling efficiencies measured in experiments using ADPNP indicate that for a supercoiled DNA substrate the probability of finding a T-segment within an open enzyme clamp is very high, and the same is true for bacterial DNA gyrase bound to a positively supercoiled DNA substrate. The situation with DNA gyrase bound to a negatively supercoiled DNA is probably rather different, however, as discussed above. In experiments using ATP as the cofactor, the coupling efficiencies are likely to be determined by the relative rates of T-segment entrance and clamp closure, rather than by the probability of finding a T-segment in an equilibrium population of enzyme molecules. For yeast DNA topoisomerase II bound to a relaxed DNA, the measured coupling efficiency of DNA transport upon addition of ADPNP is around % (Roca & Wang, ). This result suggests that the binding of a T-segment to the enzyme in its open-clamp conformation is rather weak under the experimental conditions (in m Tris HCl, pH , m EDTA, m KCl, m MgCl# and m-mercaptoethanol, at mC). The T-segment presumably goes in and out of the enzyme clamp in the absence of the nucleotide, and is not in contact with the enzyme most of the time. Stable binding of eukaryotic DNA topoisomerase II to DNA crossovers has been implicated by results obtained by electron microscopy (Zechiedrich & Osheroff, ), by the formation of DNA knots (Hsieh, ; Wassermann & Cozzarelli, ; Roca et al. ), and by experiments measuring the residual number of supercoils upon relaxation of a supercoiled DNA in the presence of varying amounts of yeast DNA topoisomerase II (Roca et al. ; relaxation was carried out with a type IB DNA topoisomerase in the absence of ATP so that the DNA transport activity of the type II enzyme was not manifested). It is uncertain in these measurements, however, whether a DNA crossover bound by an enzyme molecule represents a pair of G- and T-segments. Under conditions that favour crossover binding, the efficiency of DNA transport upon the addition of ADPNP is low (Roca et al. ). Furthermore, in the ADPNP-triggered decatenation of A simple molecular machine a supercoiled DNA ring singly linked to a relaxed DNA ring, yeast DNA topoisomerase II was found to preferentially bind to the supercoiled ring, which is consistent with the binding of the enzyme to a DNA crossover; this one-step DNA transport event was found to efficiently unlink the pair of rings, however, indicating that the T-segment is from the relaxed DNA ring rather than a crossover in the supercoiled ring (Roca & Wang, ). The plausible binding of type II DNA topoisomerases to crossovers that do not include a T-segment was also invoked to account for a remarkable property of these enzymes that was revealed by recent experiments (Rybenkov et al. ). It was observed that in the presence of ATP, a variety of the type II DNA topoisomerases can reduce the steady state fraction of knotted or catenated DNA molecules to levels up to two orders of magnitude below those at thermodynamic equilibrium.
. DNA relaxation by a type II DNA topoisomerase: how is the high efficiency of coupling achieved? In essence, the available data for the type II DNA topoisomerases suggest that in the relaxation of a positively supercoiled DNA by either yeast DNA topoisomerase II or E. coli DNA gyrase, the overall high efficiency of coupling of DNA transport to ATP usage is achieved by the high efficiencies of two consecutive reactions. First, the binding of two ATP molecules to a DNA-bound dimeric enzyme at a high ATP concentration almost always leads to the closure of the N-gate. Second, provided that a T-segment has already entered the enzyme clamp, the closure of the N-gate almost always leads to the transport of the trapped T-segment, first through the DNA gate and then the exit gate. The details of these and additional factors involved in the coupling of ATP usage to DNA transport are discussed in the sections below.
.. ATP hydrolysis and the closure of the enzyme clamp A key step in the ATP-triggered conformational cascade of a type II DNA topoisomerase is the dimerization of the ATPase domains of the enzyme following ATP binding. Therefore a high efficiency of coupling requires that dimerization must precede ATP hydrolysis. This is nicely accomplished by the participation of amino acid residues of both protomers in each of the ATPase site of the dimeric enzyme: ATP hydrolysis can not occur unless the N-gate is closed to form the ATPase catalytic pocket (Wigley et al. ; Tamura et al. ; Ali et al. ; O’Dea et al. ).
.. Coupling of ATPase activity to DNA binding In the presence of a high concentration of DNA, the ATPase activity of yeast DNA topoisomerase II is stimulated by about -fold (Lindsley & Wang, a). For the residue N-terminal fragment of the yeast enzyme, the ATPase activity is only marginally stimulated, by about %, by the presence of excess DNA (S. Olland and J. Wang, unpublished). Similarly, for purified E. coli DNA GyrB J. C. Wang protein, the ATPase activity is not significantly stimulated by DNA in the absence of the GyrA protein (Sugino et al. ; Sugino & Cozzarelli, ; Staudenbauer & Orr, ; Maxwell & Gellert, ). Because the binding of a DNA G- segment is likely to involve both the GyrA and GyrB regions of a type II DNA topoisomerase (see Section .), these results are consistent with the notion that stimulation of the ATPase activity of a type II DNA topoisomerase by DNA is largely through the binding of the enzyme to the G-segment. The binding of the two halves of a dimeric enzyme molecule to a contiguous DNA segment may facilitate dimerization of the N-gate jaws to form the active form for ATP hydrolysis; allosteric changes within the ATPase domain, triggered by the binding of a dimeric enzyme to DNA, may further enhance the ATPase activity (see the section below). Whether the DNA dependence of the ATPase activity involves interaction between the enzyme and the T-segment as well is less clear. Studies with E. coli DNA gyrase showed that the stimulation of its ATPase activity by DNA is dependent on the length of the DNA; DNA shorter than bp can stimulate the ATPase only at a high concentration, suggesting that DNA must bind to two or more sites of the enzyme to have an effect on the ATPase activity (Maxwell & Gellert, , ). These data can not distinguish, however, between a model in which multiple sites are involved in the binding of the G-segment, and one in which the multiple sites represent both G- and T-segment binding sites (Maxwell & Gellert, , ). Following the solution of the three-dimensional structure of the -kDa E. coli GyrB fragment (Wigley et al. ), site-directed mutagenesis within this fragment was carried out to replace Arg- by a glutamine (Tingey & Maxwell, ). Arg- is situated at a constriction of the channel formed by the closing of the N-gate (Fig. ), and was postulated to be a part of a binding surface for the T-segment (Wigley et al. ). Interestingly, the RQ mutant enzyme retains the intrinsic ATPase activity of the wild-type enzyme but the activity is not stimulated by DNA (Tingey & Maxwell, ). These observations led to the suggestion that the binding of the T-segment within the enzyme clamp may be required for stimulation of the ATPase activity. The effect of the RQ mutation on the DNA dependence of the ATPase activity could also be attributed to, however, an allosteric change within the ATPase clamp of the enzyme when a wild type but not an RQ mutant enzyme binds to a G-segment (see Section ..). It might seem ideal for a very high efficiency of coupling between ATP usage and DNA transport if ATP hydrolysis requires the entrance of a T-segment into a G-segment bound enzyme. This is not so. As described earlier, it appears that at a high concentration of ATP a G-segment-bound enzyme clamp may often close without capturing a T-segment. In such a situation, if the binding of a T-segment is indeed a prerequisite for ATP hydrolysis, an enzyme bound to a G-segment would get stuck in the closed clamp conformation whenever it fails to capture a T-segment upon closure of the N-gate: it would have to rely on its DNA-independent ATPase activity to free itself from the closed-clamp conformation. A simple molecular machine
.. Structural changes in the enzyme upon closure of the N-gate Following ATP binding and the closure of the N-gate, a conformational cascade ensues to effect the transport of the T-segment through the G-segment. Several structural changes in the enzyme have been detected upon the binding of ADPNP, the most conspicuous of which being the switch of an SV endoproteinase cleavage site in yeast DNA topoisomerase II (Lindsley & Wang, ). In the absence of ADPNP, this proteinase cleaves the yeast enzyme after Glu- (site A in Fig. ) and around residue (site C in Fig. ); in the presence of ADPNP, cleavage occurs after Glu- (site B in Fig. ) and site C. Limited proteolysis of the -kDa N-terminal E. coli gyrase B-subunit also showed that in the presence of ADPNP, cleavage by trypsin occurs after Lys-, but the -kDa N-terminal product is resistant to further cleavage that would occur in the absence of the nucleotide (Ali et al. ). Probing the chemical reactivity of individual yeast DNA topoisomerase II lysyl residues showed that ADPNP binding affects lysine citraconylation at at least six sites. Three of these sites, around Lys-\, \, and \, showed a reduced level of citraconylation in the presence of ADPNP (Li & Wang, ). Based on the crystal structure of the ATPase domain of E. coli gyrase and amino acid sequence alignment of type II DNA topoisomerases, it was suggested that the Lys-\ and \ sites are probably contacted directly by a bound ADPNP, and reactivity at the Lys-\ site is probably affected by ADPNP because of its location at the N-gate dimerization interface. Citraconylation at Lys-, , and , on the other hand, was found to be enhanced by ADPNP binding, suggesting that these lysyl residues become more exposed to the solvent through allosteric conformational changes induced by the binding of the nucleotide to the ATPase catalytic sites. ADPNP binding also affects reactivity of one or more lysyl residues around positions and in a less direct way: these positions are protected against citraconylation by DNA binding, and this DNA- mediated protection appears to be reduced in the presence of the non-hydrolysable ATP analogue (Li & Wang, ).
.. ATP binding\hydrolysis and DNA cleavage There are conflicting hints on the relation between the binding and hydrolysis of ATP by a type II DNA topoisomerase and the cleavage and rejoining of the G- segment. Two lines of evidence suggest that ATP utilization affects transient cleavage of DNA mediated by type II DNA topoisomerases and vice versa. First, covalent adduct formation between DNA and type II DNA topoisomerases, as revealed by the addition of a protein denaturant to the DNA–enzyme complexes, is often enhanced by the presence of ATP or ADPNP (Sugino et al. ; Peebles et al. ; Fisher et al. ; Sander & Hsieh, ; Kreuzer & Alberts, ; Osheroff, ). Second, when the active site tyrosine Tyr- of yeast DNA topoisomerase II is mutated to phenylalanine, and thus abolishing the DNA cleavage activity of the enzyme, the DNA-dependent ATPase activity of the enzyme was found to be largely abolished (W. Li, J. Roca, and J. Wang, J. C. Wang unpublished; J. Lindsley, personal communication; S. Gasser, personal communication; A. Maxwell, personal communication). In contrast, there have also been ample examples that the type II DNA topoisomerases and their truncation derivatives can cleave DNA in the absence of ATP (see for examples, Sander & Hsieh, ; Osheroff, ; Reece & Maxwell, a). Furthermore, enzymes lacking the ATPase activity because of the presence of point mutations or deletions were nevertheless found to have DNA cleavage activities similar to that of the wild-type enzyme (Jackson & Maxwell, ; Lindsley & Wang, a; Berger et al. ; O’Dea et al. ; Tingey & Maxwell, ). These seemingly conflicting observations are yet to be placed in the same mechanistic framework. It is noteworthy, however, that the experimentally measured efficiency of DNA cleavage by a type II DNA topoisomerase probably represents a composite of contributions from different conformations of the enzyme. The effect of ATP or ADPNP binding on DNA cleavage by the enzyme is probably realized only if the enzyme is in the proper closed-clamp conformation. In terms of the coupling between ATP usage and DNA transport, the effect of the nucleotide cofactor on the cleavage of the G-segment by the enzyme in its closed- clamp conformation is likely to be the important one, as the DNA transport event presumably occurs only inside the enzyme in its closed-clamp conformation. Judging from the large drop in the ATPase activity of yeast DNA topoisomerase II upon mutating the active site tyrosyl residue Tyr- to phenylalanine, the binding and\or hydrolysis of ATP is likely to have a significant effect on the cleavage of the G-segment in the closed-clamp conformation of the enzyme.
.. Entrapment of the T-segment and opening of the DNA gate Closely related to the question on the effects of ATP binding and hydrolysis on the cleavage of the G-segment is whether the cleavage reaction involves the T- segment. Studies with a bp DNA showed that the efficiency of Drosophila DNA topoisomerase II-mediated cleavage of this short DNA has a sigmoidal dependence on the concentration of the DNA (Corbett et al. ). Based on this observation, the authors suggested that the enzyme might interact with more than one -mer in its cleavage of one of them, and that the presence of a T-segment might be required for the cleavage of a G-segment (Corbett et al. ). Studies of DNA cleavage by the closed clamp form of yeast DNA topoisomerase II show however, that the transient breakage of the G-segment does not require the entrapment of a T-segment (Roca & Wang, ). In one experiment, yeast DNA topoisomerase II was first converted to the closed clamp conformation by the addition of ADPNP, and a linear DNA was allowed to thread through the enzyme annulus to provide a G-segment. When etoposide and SDS were sequentially added to the enzyme–DNA mixture, it was found that the G-segment was efficiently cleaved by the enzyme. Because only one DNA double helix can be threaded through an enzyme in its closed-clamp conformation, the finding of this experiment demonstrates that the entrapment of a T-segment is not required for the transient cleavage of the G-segment (Roca & Wang, ). The opening of the DNA gate by a type II DNA topoisomerase, however, A simple molecular machine requires not only breakage of the DNA strands through transesterification between the enzyme and DNA, but also the movement of the enzyme-linked DNA ends away from each other to permit the passage of the T-segment. These two steps are akin to unlocking a gate and opening it. Whereas the T-segment is not required for unlocking the DNA gate, its entrapment may be strongly coupled to opening it. From the crystal structure of the ATPase subfragment of bacterial gyrase B-subunit, the dimension of the archway formed by the dimerized fragment is not sufficiently large to accommodate a duplex DNA (Tingey & Maxwell, ). Therefore steric repulsion is likely to accompany the trapping of a T-segment, and this repulsion may force the widening of the DNA gate (Berger et al. ). In one experiment with the YF mutant of yeast DNA topoisomerase II, it was found that closure of the N-gate of a G-segment-bound mutant enzyme would occur normally upon the addition of ADPNP, but this closure could not trap a second DNA ring present at a high concentration (J. Roca, W. Li, and J. Wang, unpublished). This finding suggests that the widening of the DNA gate may be coupled to the trapping of the T-segment. For the mutant enzyme, unlocking and opening the DNA gate are not possible, and hence the G-segment- bound enzyme can not assume the closed clamp conformation even if a T-segment has entered the enzyme clamp. In a functional enzyme, on the other hand, widening the DNA gate upon entrapment of a T-segment allows the T-segment to move through the DNA gate into the large cavity on the other side of the G- segment. The positive surface charges lining this cavity is likely to provide further attraction for the T-segment to move into it.
.. Closure of the DNA gate and exit of the T-segment Once the T-segment has passed the G-segment and entered the large hole lined with positive surface charges (structure in Fig. ), it would seem to be in a favourable micro-environment. The question is then what drives its subsequent exit? The answer to the above question is likely to lie in the energetics of the enzyme- DNA complex in which the G-segment is cleaved and the DNA gate is wide open. This state is most likely thermodynamically unstable; the enzyme–DNA complex is probably driven into this state only when the T-segment is trapped in the closed N-terminal jaws of the protein. Therefore once the T-segment has passed through the open DNA gene into the large hole, the DNA–enzyme complex would resume its more stable conformation by closing the DNA gate. The movement of the two halves of the enzyme–DNA complex toward each other would reduce the size of the large hole, which could in turn force the exit of the T-segment (Berger et al. ). From the above discussion, it is clear that the stepwise movement of a T- segment through the multiple ports and locks of the canal in a G-segment-bound enzyme requires the existence of energetically well-balanced conformational states of the various enzyme–DNA complexes. The existence of these conformational states is in turn dependent on the presence of intricately balanced structural J. C. Wang elements or hinges in the enzyme–DNA complexes. For example, based on the crystal structures of the -kDa yeast DNA topoisomerase II fragment and the -kDa bacterial gyrase fragment, it was suggested that conformational changes at a pair of hinge structures in a type II DNA topoisomerase may lead to the opening of the C-gate for the exit of the T-segment, as illustrated in Fig. (Morais Cabral et al. ).
. Directionality of DNA transport: why is bacterial gyrase unique? Among the type II DNA topoisomerases, bacterial DNA gyrase is unique in two respects. First, whereas all type II DNA topoisomerases are ATP dependent, only DNA gyrase is capable of catalysing the negative supercoiling of DNA. Second, DNA gyrase and a derivative of it lacking the N-terminal half of the B-subunit possess an ATP-independent DNA relaxation activity, which differs from the ATP-dependent DNA transport activities of the other type II enzymes in that it is specific for negatively supercoiled DNA (Sugino et al. ; Gellert et al. , ; Higgins et al. ). [An early report also ascribed to bacterial DNA gyrase an ADPNP-dependent activity capable of removing positive supercoils catalytically, but recent measurements showed that positive supercoil removal in the presence of the non-hydrolysable analogue is stoichiometric and amounts to a maximum of two supercoils per gyrase molecule (Bates et al. ).]
... Structural basis of the ability of bacterial DNA gyrase to catalyse the ATP-dependent negative supercoiling of DNA What might be the structural features that set gyrase apart from the other members of the type II subfamily of DNA topoisomerases? It was suggested many years ago that the ability of DNA gyrase to catalyse DNA supercoiling is a consequence of the unique way DNA gyrase binds to DNA (Wang, ). Only in the case of DNA gyrase the G-segment is wrapped around the enzyme with a right-handed writhe, and about bp of the segment are in contact with the enzyme (Liu & Wang, a, b; Klevan & Wang, ; Fisher et al. ; Kirkegaard & Wang, ; Morrison & Cozzarelli, ; Rau et al. ; Orphanides & Maxwell, ); for the other type II DNA topoisomerases, a much shorter region of the G-segment (about – bp) is contacting the enzyme without a significant writhe of the segment (Spitzer & Muller, ; Lee et al. ; Thomsen et al. ). How can the wrapping of a DNA segment around a type II DNA toposiomerase affect the outcome of the enzyme-mediated topological transformation of a DNA ring? When a T-segment enters the interior of an enzyme bound to a G-segment on the same DNA ring, the two DNA segments can form a crossover with either a plus sign or a minus sign, as illustrated in Fig. . The enzyme-mediated transport of the T-segment through the G-segment always inverts this nodal sign. Therefore, in order for a type II DNA topoisomerase to catalyse the negative supercoiling of a DNA ring, the T- and G-segment must form a plus node before the DNA transport event; the linking number of the DNA ring is then reduced A simple molecular machine
(a)
GT
(b) (c)
–
+
(d)
–
+
Fig. . The sign of the node between a pair of G and T-segments in a DNA ring before the DNA transport event. The schematic in (a) illustrates a G-segment-bound enzyme (circle with a cross inside) viewed down the molecular dyad from the N-gate to the C-gate. The T- segment can enter the opened N-gate to form a positive node (b) or a negative node (c) with the G-segment. If the enzyme does not impose a significant writhe in the G-segment, the nodal sign between the incoming T-segment and the enzyme-bound G-segment must be determined by the topology of the DNA ring: it would be positive in a positively supercoiled DNA, negative in a negatively supercoiled one, and either positive or negative in a relaxed DNA ring. In drawing (d), the two crossing-segments are shown to be perpendicular to each other; the plus and minus mode drawn are structurally equivalent. In the case of bacterial DNA gyrase, the enzyme imposes a right-handed writhe in the G- segment. The nodal sign is then influenced by two terms: enzyme–DNA interaction favours a positive node, and DNA conformation favours a particular nodal sign depending on the sense of supercoiling. Illustration taken from Roca & Wang (). by two by the inversion of the plus node to a minus one. The wrapping of a DNA segment around gyrase apparently imposes a strong bias in favour of the formation of a plus node between the two segments. For the other type II DNA topoisomerases, wrapping of the DNA around the enzyme is not involved, and the nodal sign an incoming T-segment makes with the G-segment is specified by the conformation of the DNA: the segments would preferentially assume a plus sign J. C. Wang in a positively supercoiled DNA and a minus sign in a negatively supercoiled DNA (Roca & Wang, ). Thus for type II DNA topoisomerases other than gyrase, enzyme-mediated transport of a T-segment through a G-segment always reduces the net number of supercoils in the DNA. Recent studies have provided strong evidence in support of the above interpretation. A C-terminal -kDa fragment of E. coli DNA gyrase A-subunit (residues –) was shown to be involved in the right-handed wrapping of DNA around the enzyme: mutant gyrase lacking this -kDa domain does not impose a right-handed writhe in the DNA segment bound to it (Kampranis & Maxwell, ), but the purified -kDa protein does by itself (Reece & Maxwell, a). In addition, deletion of this domain from DNA gyrase converts the enzyme from a DNA-negative supercoiling activity to one that catalyses the ATP- dependent removal of supercoils (Kampranis & Maxwell, ). Unlike DNA gyrase but similar to the other type II DNA topoisomerases, this deletion mutant of gyrase is also efficient in unlinking DNA catenanes in the presence of ATP (Kampranis & Maxwell, ).
.. The ATP-independent DNA relaxation activity of gyrase The ATP-independent DNA relaxation activity of gyrase is of particular mechanistic interest in that it is specific for negative supercoils. In other words, in the absence of ATP the enzyme retains a directionality in its transport of a DNA segment through another segment of the same DNA ring, but this directionality is the opposite of that in the presence of ATP. One interpretation of the observed directionality of gyrase-mediated DNA transport in the absence of ATP is that the ATP-independent reaction follows a reverse pathway of the ATP-driven reaction: the T-segment enters through the C-gate, passes through the G-segment, and exits the N-gate (Kampranis & Maxwell, ; Cullis et al. ; Critchlow et al. ). This interpretation implies that the C-gate of DNA gyrase might not be tightly closed and might often pop open to allow a T-segment to slip through. There is no data to suggest, however, that interaction across the A–A dimer interface in DNA gyrase is weaker than that in yeast DNA topoisomerase II. The A subunit of E. coli as well as Micrococcus luteus DNA gyrase exists as stable dimers in solution under a wide range of conditions (see for examples, Klevan & Wang, ; Kirschhausen et al. ), and the crystal structure of the -kDa gyrase A fragment shows an A–A # dimer interface burying an area of over AH (Morais Cabral et al. ). A second problem with the reverse-path interpretation of the ATP-independent DNA relaxation activity of DNA gyrase is that it provides no satisfactory explanation for the specificity of the reaction, namely only negatively supercoils are removed. This problem can be better seen by first considering a gyrase molecule bound to a G-segment in a supercoiled DNA. As discussed before, the wrapping of the G-segment around the enzyme positions a T-segment, T", which makes a positive node with the G-segment, and is presumably located close to the normal entrance gate of the enzyme. This means that the T-segment which would follow the reverse path could not be this well-positioned T" segment and must A simple molecular machine therefore be a different T-segment T#. The specificity for the removal of negative supercoils would then require that the enzyme could somehow position T" and T# near the normal entrance and exit gate of the enzyme, respectively, and that the nodal sign between the G-segment and T# must be a negative one. There is presently no structural hint of such an arrangement. The above discussion demonstrates that alternatives to the reverse-path interpretation should be considered. One model that is consistent with the available data is that the ATP-independent removal of every two negative supercoils involves the binding of a DNA gyrase molecule at a negative node in a negatively supercoiled DNA, perhaps through the assembly of one GyrA dimer and two GyrB monomers at that node, to form an enzyme–DNA complex similar to the one which is formed in the normal ATP-driven pathway immediately after the passage of the T-segment through the DNA gate. In this complex, one of the arms of the negative DNA node forms the G-segment, and the other arm, which would subsequently serve as the T-segment, is present inside the cavity bounded by the A protomers and the G-segment. In the absence of the ATP dependent closure of the entrance gate and the conformational cascade which would normally follow, this pre-assembled T-segment could not pass through the normal exit gate (the C-gate). It could escape through the DNA gate whenever the gate opens, however, to form the more stable enzyme-DNA complex in which the T-segment assumes a positive-noded orientation relative to the G-segment. This process would remove two negative supercoils. In the mechanism postulated above, thermodynamic considerations predict that the formation of such a pre-assembled complex with a negative node between the two DNA segments could occur only in a negatively supercoiled DNA. Assembly of gyrase subunits at a positive node in a positively supercoiled DNA could occur, of course, but the energetics would strongly favour the formation of the normal enzyme-DNA complex before the transport of the T-segment, in which the DNA wraps around the enzyme to position a T-segment in a positive-noded orientation with respect to the G-segment. Such a thermodynamically stable complex can not be relaxed further in the absence of ATP, which explains why the ATP- independent DNA relaxation activity of bacterial gyrase does not remove positive supercoils.
. Biochemical, genetic, and crystallographic studies of the type II DNA topoisomerases in the past two decades have led to a detailed model on how such an enzyme accomplishes its remarkable feat of moving one DNA duplex through another. A type II DNA topoisomerase is in every way an ATP-fuelled molecular machine. It is now well recognized that in the biological world most if not all processes are carried out by intricate macromolecular machines, usually composed of a complex assembly of components (see for examples, Alberts, , ; Echols, ). The type II DNA topoisomerases are unusual in that they act as single molecules: a single dimeric enzyme does all the work of binding to one J. C. Wang DNA double helix, capturing a second, opening a gate in the first DNA segment, and transporting the captured segment through the transiently opened DNA gate, all in a split second! The beauty of this simple and yet efficient machine is awesome. Because of their simplicity, the type II DNA topoisomerases also provide opportunities for deeper understanding of coupled processes; their indispensability in cells and their importance as targets of antimicrobial and anticancer therapeutics add yet another bonus to their studies.
. I am grateful to many who made the study of DNA topoisomerases a joyful undertakening, to Qiyong Liu for his help in the preparation of several of the figures, to Janet Lindsley and James Berger for sharing results prior to publication, to Tao-shih Hsieh and Tony Maxwell for discussions, and to NIH for three decades of support of the research of my laboratory on DNA topoisomerases. I thank Richard Henderson and Kurt Wu$ thrich for their patience and prodding during the writing of this review; without patience they would have given up on this review, and without their prodding I would have abandoned my charge.
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