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.     

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.     

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.     

Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level.

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion; understanding the mechanical properties of these molecules helps us understand how they function in the cell. In this review, we summarize the application of magnetic tweezers (MT) to the study of DNA behavior at the single-molecule level. MT can be conveniently used to stretch DNA and introduce controlled levels of superhelicity into the molecule and to follow to a high definition the action of different types of topoisomerases. Its potential for chromatin studies is also enormous, and we will briefly present our first chromatin results.     

Single-molecule perspectives on helicase mechanisms and functions

Helicases are a diverse group of molecular motors that utilize energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to unwind and translocate along nucleic acids. These enzymes play critical roles in nearly all aspects of nucleic acid metabolism, and consequently, a detailed understanding of helicase mechanisms at the molecular level is essential. Over the past few decades, single-molecule techniques, such as optical tweezers, magnetic tweezers, laminar flow, fluorescence resonance energy transfer (FRET), and DNA curtains, have proved to be powerful tools to investigate the functional properties of both DNA and RNA helicases. These approaches allow researchers to manipulate single helicase molecules, perturb their free energy landscape to probe the chemo-mechanical activities of these motors, and to detect the conformational changes of helicases during unwinding. Furthermore, these techniques also provide the capability to distinguish helicase heterogeneity and monitor helicase motion at nanometer spatial and millisecond temporal resolutions, ultimately providing new insights into the mechanisms that could not be resolved by ensemble assays. This review outlines the single-molecule techniques that have been utilized for measurements of helicase activities and discusses helicase mechanisms with a focus on functional and mechanistic insights revealed through single-molecule investigations in the past five years.     

Analysis of the eukaryotic topoisomerase II DNA gate: a single-molecule FRET and structural perspective

Type II DNA topoisomerases (topos) are essential and ubiquitous enzymes that perform important intracellular roles in chromosome condensation and segregation, and in regulating DNA supercoiling. Eukaryotic topo II, a type II topoisomerase, is a homodimeric enzyme that solves topological entanglement problems by using the energy from ATP hydrolysis to pass one segment of DNA through another by way of a reversible, enzyme-bridged double-stranded break. This DNA break is linked to the protein by a phosphodiester bond between the active site tyrosine of each subunit and backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this enzymatic cleavage/religation of the backbone. This reversible DNA cleavage reaction is the target of a number of anticancer drugs, which can elicit DNA damage by affecting the cleavage/religation equilibrium. Because of its clinical importance, many studies have sought to determine the manner in which topo II interacts with DNA. Here we highlight recent single-molecule fluorescence resonance energy transfer and crystallographic studies that have provided new insight into the dynamics and structure of the topo II DNA gate.     

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.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

Separation and functional analysis of eukaryotic DNA topoisomerases by chromatography and electrophoresis

DNA topoisomerases are enzymes that control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently, these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. In this review we summarize current protocols for extracting and purifying DNA topoisomerases, and for separating subtypes and isoforms of these enzymes. Furthermore, we discuss methods for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumor drugs in cell-free assays.     

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria

Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.     

Holoenzyme assembly and ATP-mediated conformational dynamics of topoisomerase VI

Type II topoisomerases help disentangle chromosomes to facilitate cell division. To advance understanding of the structure and dynamics of these essential enzymes, we have determined the crystal structure of an archaeal type IIB topoisomerase, topo VI, at 4.0-A resolution. The 220-kDa heterotetramer adopts a 'twin-gate' architecture, in which a pair of ATPase domains at one end of the enzyme is poised to coordinate DNA movements into the enzyme and through a set of DNA-cleaving domains at the other end. Small-angle X-ray scattering studies show that nucleotide binding elicits a major structural reorganization that is propagated to the enzyme's DNA-cleavage center, explaining how ATP is coupled to DNA capture and strand scission. These data afford important insights into the mechanisms of topo VI and related proteins, including type IIA topoisomerases and the Spo11 meiotic recombination endonuclease.     

Honey, I shrunk the DNA: DNA length as a probe for nucleic-acid enzyme activity

The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single- and double-stranded DNA can be used as a probe to study the dynamics of these enzymes at the single-molecule level.     

A highly processive topoisomerase I: studies at the single-molecule level

Amongst enzymes which relieve torsional strain and maintain chromosome supercoiling, type IA topoisomerases share a strand-passage mechanism that involves transient nicking and re-joining of a single deoxyribonucleic acid (DNA) strand. In contrast to many bacterial species that possess two type IA topoisomerases (TopA and TopB), Actinobacteria possess only TopA, and unlike its homologues this topoisomerase has a unique C-terminal domain that lacks the Zn-finger motifs characteristic of type IA enzymes. To better understand how this unique C-terminal domain affects the enzyme''s activity, we have examined DNA relaxation by actinobacterial TopA from Streptomyces coelicolor (ScTopA) using real-time single-molecule experiments. These studies reveal extremely high processivity of ScTopA not described previously for any other topoisomerase of type I. Moreover, we also demonstrate that enzyme processivity varies in a torque-dependent manner. Based on the analysis of the C-terminally truncated ScTopA mutants, we propose that high processivity of the enzyme is associated with the presence of a stretch of positively charged amino acids in its C-terminal region.     

Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193.     

High-throughput assays for DNA gyrase and other topoisomerases

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.