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Cassidy Crook Within the late 1960’s and early 1970’s the disciplines of biochemistry and molecular biology were wrought with questions pertaining to the double helical structure of DNA, as elucidated by J. D. Watson and F. Crick (1953). Of considerable interest at this time was the newfound idea of whole chromosome-sized DNA molecules; that is, that one molecule of DNA might comprise an entire chromosome (Kavenoff et al, 1973). Evidence that DNA molecules were far longer than previously believed led to the recognition of a DNA replication problem, first proposed by M. Delbrück (Delbrück, 1979; Holmes, 1998). Delbrück argued that strand separation of the DNA double helix during the process of replication would introduce considerable strain to the helix, inducing a supercoiled state. Indeed, studies of polyoma viral DNA in the 1960’s led to the discovery of the phenomenon of DNA supercoiling on the basis of observed anomalous fractionation patterns of covalently-closed circular DNA (Bates & Maxwell, 2005). DNA supercoiling is the result of topological strain on the DNA helix caused by over- or under-winding of DNA. Two distinct [topo]isomeric forms of supercoiled DNA exist: positively and negatively supercoiled DNA. Positive supercoiling is the consequence of over-twisting the DNA helix, while negative supercoiling results from helix unraveling. Central in considering the topological characteristics of deformed DNA is the quantitative parameter linking number (Lk), which essentially describes the number of helical turns in a DNA molecule (i.e., the number of linkages between the two strands). Because the helical structure of DNA is an intrinsic property of its chemical composition, a standard Lk° can be determined for a DNA molecule with a length N, such that Lk°= N/10.5, where 10.5 is the number of base pairs per helical turn of DNA under standard biological conditions. Thereby, negatively supercoiled DNA is any in which Lk is less than Lk° and likewise, Lk is greater than Lk° describes positively supercoiled DNA (Bates & Maxwell, 2005; Wang, 1998). It is evident that a host of topological considerations influence the myriad cellular processes involving DNA. Therefore, it comes as no surprise that DNA topology is highly regulated through catalytic protein interactions. Specifically, the interconversion of isomeric DNA topologies is facilitated by the family of enzymes called DNA topoisomerases (topos). In a general sense, topoisomerases catalyze the interpenetration of DNA strands or individual DNA molecules, indirectly coupled to the energy of ATP hydrolysis or the energy of supercoiling itself (Wang, 1998). Topoisomerase was first purified from E. coli by J.C. Wang (1971) and coined ω. In his pioneering
work, Wang observed that incubation of ω with negative superhelical DNA reduced the number of superhelical turns (Wang, 1971). Today ω protein is known as topo IA (Corbett & Berger, 2004). Topoisomerase enzymes comprise a diverse protein superfamily with multiple constituents found in all organisms, from mammals to plants to archaea, as well as certain viruses. Two primary classes of topoisomerases are defined: type I and type II enzymes. These two categories of topos differ in three distinct respects: (a) type I enzymes are solely capable of single strand cleavage, whereas type II enzymes catalyze double strand cleavage (corresponding to a discrete change in Lk of ±2/catalytic cycle, for type II). (b) The catalytic mechanism of type I enzymes is independent of ATP, while type II enzymes are ATP-dependent (Corbett & Berger, 2004). (c) Type I topos are capable of knotting/unknotting circular ssDNA, catalyzing circular duplex formation, and catenation/decatenation of nicked DNA circles, whereas type II enzymes are capable of knotting/unknotting circular dsDNA, and inducing catenation/decatenation between intact circular duplexes (Bates & Maxwell, 2005). Topoisomerases are further divided into subfamilies IA, IB, IIA, and IIB. Type IA enzymes include eubacterial topoI and topoIII, yeast topoIII, mammalian topoIIIα and β, and reverse gyrase. Nearly all members of this subfamily exhibit monomeric subunit architecture (excluding M. kandleri reverse gyrase). Furthermore, IA topos are mechanistically defined by the stabilization of strand cleavage through the attachment of a 5' phosphate (as opposed to a 3' phosphate) to an active site tyrosine residue, dependence on Mg(II), and exclusive substrate specificity for negative supercoils. Comparatively, examples of type IB enzymes include eukaryotic topoI, Pox virus topo, and eubacterial topoV. The type IB subfamily differs from type IA in that no metal cofactor is required, both negative and positive supercoils can serve as substrates, and strand cleavage is stabilized by covalent attachment of a 3' phosphate to an active site tyrosine (Corbett & Berger, 2004). In contrast to type I topos, the type IIA subfamily comprise nearly all type II topos, with the sole exception of type IIB archaeal topoVI. IIA enzymes include eubacterial DNA gyrase (Gyr), eubacterial topo IV, yeast topoII, and mammalian topoIIα and β. DNA Gyrase is of particular interest due to its ability to induce negative supercoiling, in addition to relaxing supercoils. Yeast and mammalian type II topos are characterized by homodimeric subunit organization, while Gyr and type IIB topoVI are heterotetramers of the structure A2B2 and topoIV is a heterotetramer of the form C2E2 (Champoux, 2001). The cellular roles of type II topos include the release of intertwined chromosome pairs during mitosis, a consequence of their decatenation activity; interestingly, E. coli cells lacking topoisomerase cannot survive (Wang, 1998). Furthermore, type II topoisomerases
possess intriguing structural and mechanistic features underlying their distinctive function; for example, the role of ATP hydrolysis in enzyme catalysis is highly unique. Type IIA topoisomerases are comprised of three predominant functional regions consecutively joined together by flexible linkers: the amino-terminal ATPase dimeric region formed by the GHKL and transducer domains, the toprim and 5Y-CAP domains, and the C-terminal scaffolding/accessory domain (C-gate). The two ATPase N-terminal regions of the homodimeric topoII enzyme collectively form an ATP-operated clamp (N-gate) structure. The enzyme’s midregion, composed of the conserved toprim and 5Y-CAP domains, plays a central role in DNA binding and strand cleavage. The 5Y-CAP is so-called because it contains a “winged-helix” DNA binding fold found in E. coli Catabolite Activator Protein, as well as the catalytic Y(782) residue that forms a phosphodiester linkage with the DNA 5' terminus following nucleophilic attack via transesterification, hence 5Y-CAP. Additionally, an R residue adjacent to Y782 is thought to function in positioning the scissile bond. The toprim domain is so-named because this fold is found in topoisomerases as well as primases. This domain is functionally characterized by an acidic cluster thought to act in chelating the Mg2+ cofactor (Corbett & Berger, 2004). Catalysis by type II topoisomerases occurs through a double-gate strand passage reaction mechanism, in which both strands of a dsDNA segment are cleaved and a second duplex segment is passed through the break. This process may be either intramolecular, as in the case of superhelical relaxing, or intermolecular, as in the formation/deformation of catenanes. Firstly, the G (gate) segment is bound at the high affinity DNA binding folds of the CAP domains in a manner dependent on an open N-gate conformation. G segment binding induces an increase in N-gate cycling between open and closed states, corresponding to ATP binding and ADP+Pi release, until the T (transported) segment is captured (Wang, 1998). T-segment capture and N-gate closing are followed by transient cleavage of the G segment by the two homologous active site Y residues and opening of the DNA gate formed by the 5Y-CAP and scaffold domains. It has been suggested that DNA gate opening is facilitated by steric repulsion of the T segment, whereby the T segment is drawn toward the DNA gate by an electrostatic potential gradient. Indeed, quantitative analysis has demonstrated that the homodimer interface and the large cavity on the opposite side of the G segment are lined with positive charges, whereas the N-gate arms are highly acidic (Berger et al 1996). Furthermore, it is thought that migration of the T segment into the central hole results in the collapse of the DNA gate followed by processive constriction at the homodimer interface to support
re-ligation of the G segment, which subsequently causes narrowing of the central hole and forces the T segment to be expelled through the C-gate (Berger et al 1996). The GHKL and transducer domains of the N-terminal ATPase dimeric region are conserved among type IIA and type IIB topoisomerases. The GHKL fold facilitates ATP binding and hydrolysis. This domain is named for the host of proteins in which it is known to occur: DNA Gyrase, Hsp90, CheA-family histidine Kinases, and MutL. The secondary structure of the ATP binding site is comprised of a “floor” of β-sheets enclosed by several α-helices, as well as a glycine rich P-loop lid structure (Corbett & Berger, 2004). ATP binding induces dimerization of the GHKL domains via the dual interaction of an N-terminal strap motif from one chain with the GHKL Ploop on the opposite chain. Thus, the ATP-operated clamp adopts a closed conformation upon ATP binding through the dimerization of GHKL domains. Coordination of the ATP-lid (i.e., Ploop) by the GHKL strap motif on the adjacent monomer effectively positions the ATP γphosphate for hydrolysis. Importantly, mutagenesis studies have identified a critical lysine residue (Gyr K337) in ATP hydrolysis that is capable of hydrogen bonding with the γ-phosphate, potentially suggesting its involvement in transition state stabilization (Smith & Maxwell, 1998). Is it thought that a rotation between the GHKL and transducer domains is prompted by ATP binding and GHKL dimerization, facilitating the positioning of lysine within the GHKL active site (Corbett & Berger, 2004). Moreover, mutagenesis studies have led to a proposed mechanism for ATP hydrolysis in DNA gyrase, whereby Glu42 acts as a general base and is aligned by His38 in a phosphotransfer reaction mediated by water (Jackson & Maxwell, 1993). The type II topoisomerase ATPase reaction cycle proceeds sequentially, such that the enzyme binds two ATP molecules, with one ATP being rapidly hydrolyzed. After the rapid hydrolysis of one ATP, the T segment is transferred through the DNA gate, followed by the ratedetermining release of ADP and Pi, and ultimately the hydrolysis and release of the second ATP (Baird et al 2001). Although the mechanism of ATP binding and hydrolysis has been fairly well detailed, the structural means by which the ATPase reaction triggers the conformational changes that facilitate strand passage remain unclear. However, it is thought that the rotation of the GHKL and transducer domains in positioning of the ATPase catalytic lysine is critical in signaling the structural rearrangements that direct strand passage (Corbett & Berger, 2004). Furthermore, while it is clear that the ATPase reaction cycle plays a critical role in the conformational mechanism of type II topoisomerases, the basis for coupling the free energy of ATP hydrolysis to the exergonic reaction
of DNA relaxation is not so apparent. In contrast, the role of ATP in the endergonic negative supercoiling mechanism of the type IIA bacterial topo, DNA gyrase, is considerably more intuitive. DNA gyrase (Gyr) is a unique type IIA topoisomerase due to its characteristic ability to introduce negative supercoils into DNA. The ability of Gyr to under-wind DNA is functionally significant in bacteria, as it decreases the energy required for DNA melting in critical processes, such as replication and transcription (Zechiedrich et al 1995). DNA gyrase boasts a DNA binding/cleavage region with an increase in mass of 33 kDa (Kampranis & Maxwell, 1999). This modification translates into the ability to wrap approximately 140 base pairs of G segment DNA around the entire enzyme with a right-handed writhe (Champoux, 2001). Through a mechanism that is not well understood, wrapping of the G segment enables Gyr to distinguish between a negative and a positive node formed between the T and G segments and selectively construct a positive node, such that a negative node is formed by segment transfer (Kampranis & Maxwell, 1999). As one might expect, ATP hydrolysis is critical in the Gyr wrapping reaction, exemplified by the observation that binding of the non-hydrolyzable ATP analog, ADPNP, destabilizes wrapping (Heddle et al 2004). In addition, it has been observed that the supercoiling reaction of Gyr reaches a limit, despite the persistence of ATP hydrolysis. This limit is explained in terms of the influence of substrate topology on strand passage, whereby strand passage is efficiently coupled to ATP hydrolysis in the presence of a positively supercoiled substrate and coupling is destabilized in the presence of a moderately negative supercoiled substrate. However, because ATP hydrolysis is foremost dependent on the trapping of the T segment, not strand passage, the enzyme will continue to hydrolyze ATP despite the topological nature of the substrate (Bates & Maxwell, 2007). However, dissimilar from the supercoiling reaction of DNA gyrase, DNA relaxation by type II topoisomerases presents an apparent incongruity between the energetically favorable nature of DNA relaxation and the dependence of this reaction on ATP hydrolysis. This kinetic conundrum was not resolved until 1997 by V. V. Rybenkov et al. Previously, the conventional notion of type II topos was such that these enzymes indiscriminately acted on DNA molecules to generate a population of DNA topologies (supercoils, knots, and catenanes) at an equilibrium distribution. It was believed that topos effectively facilitated topological equilibrium by figuratively “converting real DNA molecules into phantom chains that freely pass through themselves” (Vologodskii, 1998). However, the work of Rybenkov et al demonstrated that, in actuality, topoisomerase II enzymes do not act arbitrarily on DNA molecules, but rather serve to control global topology favoring topological simplification – coined the “Rybenkov effect”. Specifically, it was demonstrated that the
steady state levels of DNA catenanes and knots were as much as 80-fold lower than at thermodynamic equilibrium (Rybenkov et al 1997). Therefore, the free energy of ATP hydrolysis is effectively utilized to drive a global topological state away from equilibrium. The primary implication of the Rybenkov effect is that topoisomerase II enzymes do not merely catalyze strand passage, but are able to efficiently assess DNA topology through an unknown mechanism. Several distinct models have been proposed to account for the mechanism by which type II topos achieve a sub-equilibrium steady state topological diversity. All models currently account for topological simplification by an increase in topoisomerase specificity for T segments that would decrease topological complexity. In addition, current models fall into two broad categories: geometric and kinetic (Stuchinskaya et al 2009). The first mechanistic model for topological recognition was proposed by Rybenkov et al (1997). This model suggests the existence of a third binding site on topo, resulting in the formation of a crossover complex and potentially facilitating non-random selection of a T segment by movement of the crossover complex along the DNA. Additionally, sliding of the crossover complex would yield a cluster of nodes within a small loop, which could then be opportunistically incorporated into the enzyme complex (Rybenkov et al, 1997). However, this model is thought to be implausible due to the dual role of ATP for sliding and strand passage, as well as the lack of evidence for a third DNA binding site (Maxwell et al, 2005; Vologodskii, 1998). A simplified geometric model maintains that the G segment is wrapped around the enzyme to form a hairpin structure with the topo positioned inside the hairpin loop, requiring the T segment to enter the mouth of the hairpin structure. Computer simulation experiments based on this model have indicated a steady state knot fraction 5 to 10-fold lower than equilibrium; however, experimental steady state values obtained from topo II enzymes were 5 to 100-fold lower than equilibrium (Vologodskii, 1998; Vologodskii et al, 2001). In addition, it has been proposed that this model is not applicable to large circular DNA molecules. However, recent experimental results demonstrate the persistence of the Rybenkov effect independent of circular DNA size, potentially indicating that an alternative mechanism is employed (Bates & Maxwell, 2007; Stuchinskaya et al 2009). One example of a kinetic model for explaining the Rybenkov effect is known as “kinetic proofreading”. In this model ATPase action and strand passage are uncoupled (to some degree). Uncoupling of the ATPase N-gate and T segment transfer could enable T segments to be transiently captured and selectively released. Moreover, this mechanism could function in isolation or as a compliment to a geometric model for T segment specificity (Stuchinskaya et al 2009).
Although it has been suggested that DNA topology simplification is an advantageous or adaptive process, the possibility remains that DNA hyper-relaxation is of minimal biological relevance due to the fact that DNA in vivo is under constant regulation and, therefore, likely only transiently relaxed. However, the unknotting action of TopoII enzymes may be of greater significance, as it has been suggested that knotting is associated with genetic rearrangements. Also, it is thought that topological simplification is highly inefficient based on estimates that less than 0.2 kJ mol-1 of energy is needed to drive the simplification process, while 150kJ mol-1 is liberated in the hydrolysis of two ATP. Interestingly, DNA gyrase is able to utilize most of the free energy of ATP hydrolysis through its ability to introduce negative supercoils. This may indicate a loss of function in topo IIA over evolutionary time (Stuchinskaya et al 2009). Yet, despite the inefficiency of ATP hydrolysis in the catalytic function of type IIA topos, this family of proteins serves as an essential DNA remodeling factor in all cells. Moreover, topoisomerases offer promise as potential drug targets due to their vital role in cell proliferation. Of particular interest is the development of antitumor drugs that target topos; one example is camptothecin, which acts by disrupting the enzyme at the DNA gate region (Bates & Maxwell, 2005). Evident in their essential function, diverse applications, and widespread distribution, type II topoisomerases comprise a highly sophisticated class of molecular machines that are incredible not only in their biological roles, but in the structures and mechanisms which their functions are rooted.
Literature Cited Baird, C. L. et al (2001). The ATPase reaction cycle of yeast DNA topoisomerase II. J. Biol. Chem. 276, pp.27893-27898. Bates, A. D. & Maxwell, A. (2007). Energy coupling in type II topoisomerases: why do they hydrolyze ATP? Biochem. 46, pp.7929-7941. Bates A. D. & Maxwell A. (2005) DNA topology. New York, NY: Oxford University Press, Inc. Berger, J. M. et al (1996). Structure and mechanism of DNA topoisomerase II. Nature. 379, pp. 225232. Champoux J. J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369-413. Corbett, K.D. & Berger, J.M. (2004). Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct. 33, pp.95-118. Heddle, J.G. et al (2004). Nucleotide binding to DNA gyrase causes loss of DNA wrap. J. Mol. Biol. 337, pp.597-610. Holmes, F. L. (1998). The DNA replication problem. Trends Biochem. Sci. 23, pp.117-120. Jackson, A. P. & Maxwell, A. (1993). Identifying the catalytic residue of the ATPase reaction of DNA gyrase. Proc. Natl. Acad. Sci. 90, pp.11232-11236. Kampranis, S. C. et al (1999). A model for the mechanism of strand passage by DNA gyrase. Proc. Natl. Acad. Sci. USA. 96, pp.8414-8419. Kavenoff, R., Klotz, L. C. & Zimm, B.H. (1974). On the nature of chromosome-sized DNA molecules. Cold Spring Harbor Symp. Quant. Biol. 38, 1-8. Maxwell, A. et al (2005). Coupling ATP hydrolysis to DNA strand passage in type IIA DNA topoisomerases. Biochem. Soc. Trans. 33,pp. 1460-1464. Rybenkov, V. V. et al (1997). Simplification of DNA topology beyond equilibrium values by type II topoisomerases. Science. 277, pp.690-693. Smith, C. V. & Maxwell, A. (1998). Identification of a residue involved in transition-state stabilization in the ATPase reaction of DNA gyrase. Biochem. 37, pp. 9658-9667. Stuchinskaya, T. et al (2009). How do type II topoisomerases use ATP hydrolysis to simplify DNA topology beyond equilibrium? Investigating the relaxation reaction of nonsupercoiling type II topoisomerases. J. Mol. Biol. 385, pp.1397-1408. Vologodskii, A (1998). Maxwell demon and topology simplification by type II topoisomerases. Recomb. pp.266-269.
Vologodskii, A.V. et al (2001). Mechanism of topology simplification by type II DNA topoisomerases. Proc. Natl. Acad. Sci. 98, pp.3045-3049. Wang, J. C. (1998) Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Quart. Rev. Biochem. 31, pp.107-144. Wang, J. C. (1971). Interaction between DNA and Escherichia coli protein ω. J. Mol. Biol. 55, 523-533 Zechiedrich, E. L. et al (1995). Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli. Genes Dev. 9, pp.2859-2869.
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