Single-molecule Measurements of DNA Topology and Topoisomerases

Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.     

Untangling intracellular DNA topology

The biochemical steps by which bacterial topoisomerases alter the topology of DNA are well known. However, it has been a more vexing task to establish physiological roles and sites of action of the different topoisomerases within the context of the bacterial cell cycle. This difficulty can be attributed in part to the redundancy among the activities of the different enzymes. In this microreview, we will focus on recent progress in understanding the topological structure of the chromosome, analysis of topoisomerase mechanism in single-molecule assays and recent data on the regulation and integration of topoisomerase activity within the cell cycle that have all brought a new perspective to the action of topoisomerases in the bacterial cell.     

Molecular and Structural Transactions at Human DNA Replication Origins

The DNA replication origins of metazoan genomes are the sites of complex sequence-specific protein-DNA interactions determining their precise cycle of activation and deactivation, once only along each cell cycle. Some of the involved proteins have been identified (and particularly the essential six-protein Origin Recognition complex, ORC) thanks to their homology with the proteins identified in yeast. Whereas in the latter organism ORC has a specific affinity for an origin consensus, metazoan (and human) ORC shows no sequence specificity and no origin consensus is identifiable in their genomes. The modulation of topology around the origin sequence plays an essential role in the function of the human lamin B2 origin and the two topoisomerases interact specifically with it in a cell-cycle modulated way. The two enzymes are never present on the origin at the same time and compete, in different moments of the cell cycle, with the ORC2 subunit for the same sites in the origin area. The topoisomerases could give essential contributions to origin definition, as demonstrated by their capacity to bind specifically, in vitro the lamin B2 origin, either alone (topoisomerase I) or in a multi-protein complex (topoisomerase II). They also play critical roles in the origin activation-deactivation cycle, topoisomerase II probably contributing to attain and/or maintain a topological status fit for pre-replicative complex assembly and topoisomerase I allowing the topological adaptations necessary for initiation of bi-directional synthesis.     

Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage

By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.     

Catalytic function of DNA topoisomerase II.

Although the genetic code is defined by a linear array of nucleotides, it is the three-dimensional structure of the double helix that regulates most of its cellular functions. Over the past two decades, it has become increasingly clear that aspects of this three-dimensionality which reflect topological relationships within the double helix (i.e., superhelical twisting, knotting, or tangling) influence virtually every facet of nucleic acid physiology. In vivo, DNA topology is modulated by ubiquitous enzymes known as topoisomerases. The type II enzyme is essential to the eukaryotic cell and is required for unlinking daughter chromosomes and maintaining chromosome structure. Moreover, topoisomerase II also has been identified as the primary cellular target for several widely used antineoplastic drugs. Before the physiological functions of topoisomerase II can be effectively dissected or its drug interactions fully exploited, it is imperative to understand the mechanism by which this important enzyme carries out its catalytic cycle.     

Local sensing of global DNA topology: from crossover geometry to type II topoisomerase processivity

Type II topoisomerases are ubiquitous enzymes that control the topology and higher order structures of DNA. Type IIA enzymes have the remarkable property to sense locally the global DNA topology. Although many theoretical models have been proposed, the molecular mechanism of chiral discrimination is still unclear. While experimental studies have established that topoisomerases IIA discriminate topology on the basis of crossover geometry, a recent single-molecule experiment has shown that the enzyme has a different processivity on supercoiled DNA of opposite sign. Understanding how cross-over geometry influences enzyme processivity is, therefore, the key to elucidate the mechanism of chiral discrimination. Analysing this question from the DNA side reveals first, that the different stability of chiral DNA cross-overs provides a way to locally sense the global DNA topology. Second, it shows that these enzymes have evolved to recognize the G- and T-segments stably assembled into a right-handed cross-over. Third, it demonstrates how binding right-handed cross-overs across their large angle imposes a different topological link between the topoIIA rings and the plectonemes of opposite sign thus directly affecting the enzyme freedom of motion and processivity. In bridging geometry and kinetic data, this study brings a simple solution for type IIA topoisomerase chiral discrimination.     

DNA topoisomerase II, genotoxicity, and cancer

Type II topoisomerases are ubiquitous enzymes that play essential roles in a number of fundamental DNA processes. They regulate DNA under- and overwinding, and resolve knots and tangles in the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate segment of DNA. Because type II topoisomerases generate DNA strand breaks as a requisite intermediate in their catalytic cycle, they have the potential to fragment the genome every time they function. Thus, while these enzymes are essential to the survival of proliferating cells, they also have significant genotoxic effects. This latter aspect of type II topoisomerase has been exploited for the development of several classes of anticancer drugs that are widely employed for the clinical treatment of human malignancies. However, considerable evidence indicates that these enzymes also trigger specific leukemic chromosomal translocations. In light of the impact, both positive and negative, of type II topoisomerases on human cells, it is important to understand how these enzymes function and how their actions can destabilize the genome. This article discusses both aspects of human type II topoisomerases.     

Helicase-appended Topoisomerases: New Insight into the Mechanism of Directional Strand Transfer

DNA strand passage through an enzyme-mediated gate is a key step in the catalytic cycle of topoisomerases to produce topological transformations in DNA. In most of the reactions catalyzed by topoisomerases, strand passage is not directional; thus, the enzyme simply provides a transient DNA gate through which DNA transport is allowed and thereby resolves the topological entanglement. When studied in isolation, the type IA topoisomerase family appears to conform to this rule. Interestingly, type IA enzymes can carry out directional strand transport as well. We examined here the biochemical mechanism for directional strand passage of two type IA topoisomerases: reverse gyrase and a protein complex of topoisomerase IIIα and Bloom helicase. These enzymes are able to generate vectorial strand transport independent of the supercoiling energy stored in the DNA molecule. Reverse gyrase is able to anneal single strands, thereby increasing linkage number of a DNA molecule. However, topoisomerase IIIα and Bloom helicase can dissolve DNA conjoined with a double Holliday junction, thus reducing DNA linkage. We propose here that the helicase or helicase-like component plays a determinant role in the directionality of strand transport. There is thus a common biochemical ground for the directional strand passage for the type IA topoisomerases.     

Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I: Structure guided discovery and optimization of dual targeting agents with potent,broad-spectrum enzymatic activity

The bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) are essential enzymes that control the topological state of DNA during replication. The high degree of conservation in the ATP-binding pockets of these enzymes make them appealing targets for broad-spectrum inhibitor development. A pyrrolopyrimidine scaffold was identified from a pharmacophore-based fragment screen with optimization potential. Structural characterization of inhibitor complexes conducted using selected GyrB/ParE orthologs aided in the identification of important steric, dynamic and compositional differences in the ATP-binding pockets of the targets, enabling the design of highly potent pyrrolopyrimidine inhibitors with broad enzymatic spectrum and dual targeting activity.     

Crystal structure of full length topoisomerase I from Thermotoga maritima

DNA topoisomerases are a family of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. Bacterial and archeal type IA topoisomerases, including topoisomerase I, topoisomerase III, and reverse gyrase, are crucial in regulation of DNA supercoiling and maintenance of genetic stability. The crystal structure of full length topoisomerase I from Thermotoga maritima was determined at 1.7A resolution and represents an intact and fully active bacterial topoisomerase I. It reveals the torus-like structure of the conserved transesterification core domain comprising domains I-IV and a tightly associated C-terminal zinc ribbon domain (domain V) packing against domain IV of the core domain. The previously established zinc-independence of the functional activity of T.maritima topoisomerase I is further supported by its crystal structure as no zinc ion is bound to domain V. However, the structural integrity is preserved by the formation of two disulfide bridges between the four Zn-binding cysteine residues. A functional role of domain V in DNA binding and recognition is suggested and discussed in the light of the structure and previous biochemical findings. In addition, implications for bacterial topoisomerases I are provided.     

Conformational changes in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for maintaining the topological states of DNA. In order to change the topology of DNA, topoisomerases pass one or two DNA strands through transient single or double strand breaks in the DNA phosphodiester backbone. It has been proposed that both type IA and type II enzymes change conformation dramatically during the reaction cycle in order to accomplish these transformations. In the case of Escherichia coli DNA topoisomerase I, it has been suggested that a 30 kDa fragment moves away from the rest of the protein to create an entrance into the central hole in the protein. Structures of the 30 kDa fragment reveal that indeed this fragment can change conformation significantly. The fragment is composed of two domains, and while the domains themselves remain largely unchanged, their relative arrangement can change dramatically.     

An insight into the active site of a type I DNA topoisomerase from the kinetoplastid protozoan Leishmania donovani

DNA topoisomerases are ubiquitous enzymes that govern the topological interconversions of DNA thereby playing a key role in many aspects of nucleic acid metabolism. Recently determined crystal structures of topoisomerase fragments, representing nearly all the known subclasses, have been solved. The type IB enzymes are structurally distinct from other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. A putative topoisomerase I open reading frame from the kinetoplastid Leishmania donovani was reported which shared a substantial degree of homology with type IB topoisomerases but having a variable C-terminus. Here we present a molecular model of the above parasite gene product, using the human topoisomerase I crystal structure in complex with a 22 bp oligonucleotide as a template. Our studies indicate that the overall structure of the parasite protein is similar to the human enzyme; however, major differences occur in the C-terminal loop, which harbors a serine in place of the usual catalytic tyrosine. Most other structural themes common to type IB topoisomerases, including secondary structural folds, hinged clamps that open and close to bind DNA, nucleophilic attack on the scissile DNA strand and formation of a ternary complex with the topoisomerase I inhibitor camptothecin could be visualized in our homology model. The validity of serine acting as the nucleophile in the case of the parasite protein model was corroborated with our biochemical mapping of the active site with topoisomerase I enzyme purified from L.donovani promastigotes.     

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.     

Functional interactions of DNA topoisomerases with a human replication origin

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.