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Topoisomerase

Topoisomerases are essential enzymes found in all living organisms that manage DNA topology by introducing transient single- or double-strand breaks in the DNA double helix, allowing the strands to pass through one another to relieve torsional stress, resolve supercoils, and disentangle intertwined DNA molecules during processes such as replication, transcription, and chromosome segregation.^[1] These enzymes are highly conserved across bacteria, archaea, and eukaryotes, underscoring their fundamental role in maintaining genome integrity and enabling cellular function.^[2] Topoisomerases are classified into two main types based on their mechanism of action: Type I topoisomerases, which create a single-strand break and typically relax supercoiled DNA without requiring ATP, and Type II topoisomerases, which generate double-strand breaks and use ATP hydrolysis to drive the passage of one DNA duplex through another, facilitating both relaxation and decatenation.^[1] Type IA enzymes, such as bacterial topoisomerase I and eukaryotic topoisomerase III, strand-pass a single DNA segment through a temporary nick in the opposite strand, while Type IB enzymes, like eukaryotic topoisomerase I, allow controlled rotation of the DNA around the break to unwind supercoils.^[1] In contrast, Type II enzymes are subdivided into Type IIA (e.g., DNA gyrase and topoisomerase IV in bacteria, topoisomerase II in eukaryotes) and Type IIB (e.g., topoisomerase VI in archaea and plants), with Type IIA being ubiquitous in promoting DNA supercoiling and segregation.^[2] The biological significance of topoisomerases extends to nearly every aspect of DNA metabolism, as unrestrained supercoiling or catenation can halt replication forks, impede RNA polymerase progression, and lead to chromosomal instability.^[1] For instance, Type II topoisomerases are indispensable for separating newly replicated chromosomes during mitosis and meiosis, while Type I enzymes primarily support transcription by alleviating positive supercoils ahead of the transcription machinery.^[2] Dysregulation or inhibition of topoisomerases can result in cell death or genomic mutations, highlighting their therapeutic potential in targeting rapidly dividing cells, such as in cancer treatment.^[1]

Fundamentals

Overview and Importance

Topoisomerases are enzymes that regulate DNA topology by transiently breaking one or both strands of the DNA double helix, allowing the passage of another DNA segment through the break, and then resealing the break to resolve supercoils, knots, and catenanes.^[3] These actions prevent the accumulation of topological stress that could otherwise lead to DNA damage during essential cellular activities.^[3] The biological importance of topoisomerases lies in their critical roles in facilitating DNA replication, transcription, and chromosome segregation, where they relieve torsional strain generated by the unwinding of the double helix and decatenate intertwined daughter molecules.^[4] Without topoisomerases, these processes would be severely impaired, compromising genome stability and cell viability across all domains of life, including bacteria, archaea, and eukaryotes.^[3] Topoisomerases exhibit remarkable evolutionary conservation, reflecting their fundamental necessity in managing DNA structure from prokaryotes to higher organisms; for instance, bacterial DNA gyrase introduces negative supercoils to compact the genome, while eukaryotic topoisomerase II decatenates chromosomes during mitosis.^[5] A key topological invariant they influence is the linking number (Lk), which quantifies the number of times one DNA strand winds around the other in a closed circular molecule.^[6]

DNA Topology Basics

DNA exists as a double-stranded helical molecule, and its topology is characterized by three key parameters: the linking number (Lk), which represents the number of times one strand crosses the other when the molecule is projected onto a plane; the twist (Tw), which measures the number of helical turns within the double helix; and the writhe (Wr), which quantifies the coiling of the helix axis in space.^[7] These parameters are related by the equation Lk = Tw + Wr, a topological invariant for closed circular DNA that remains constant unless the strands are broken and resealed.^[8] In relaxed B-form DNA, the linking number corresponds to approximately one turn every 10.5 base pairs, so for a DNA molecule with N base pairs, the relaxed linking number is Lk_0 = N / 10.5.^[8] Supercoiling occurs when the actual linking number deviates from this relaxed state: negative supercoiling (underwound DNA, \Delta Lk < 0) reduces the linking number, while positive supercoiling (overwound DNA, \Delta Lk > 0) increases it, leading to torsional stress that can manifest as writhe to relieve strain.^[7] Unmanaged DNA topology generates significant biological challenges, including torsional stress that hinders the unwinding required for DNA replication and transcription by impeding the progress of polymerases.^[9] Additionally, during chromosome segregation, unrestrained supercoiling can result in the formation of knots within individual DNA molecules or catenanes linking daughter strands, potentially blocking cell division.^[10] Topoisomerases address these issues by transiently breaking and rejoining DNA strands to alter the linking number, thereby relaxing supercoils or, in the case of DNA gyrase, actively introducing negative supercoils using ATP hydrolysis to maintain optimal topology for cellular processes.^[11]

Historical Development

Early Discovery

The concept of DNA supercoiling emerged in the mid-1960s through studies on bacterial plasmids, revealing that closed circular DNA molecules exist in a negatively supercoiled state that affects their sedimentation behavior in ultracentrifugation assays. This topological feature prompted investigations into enzymes that could modulate it, leading to the identification of topoisomerases. In 1971, James C. Wang discovered the first DNA topoisomerase while studying proteins involved in Escherichia coli DNA replication. He purified a protein, initially designated ω (omega), that relaxed supercoiled phage λ DNA into a slower-sedimenting relaxed form, as detected by zone sedimentation velocity in sucrose gradients and cesium chloride-ethidium bromide density gradient centrifugation. This Type I topoisomerase, now known as E. coli DNA topoisomerase I, demonstrated the ability to relieve torsional stress in DNA without requiring energy input, marking a foundational advance in understanding DNA topology management. Early genetic evidence for topoisomerase function arose from E. coli mutants with defects in the topA gene encoding topoisomerase I, isolated in the mid-1970s. These mutants exhibited excessive negative supercoiling of the chromosome and plasmids, resulting in lethality that could only be suppressed by compensatory mutations near gyrase genes, underscoring the critical balance of supercoiling for bacterial viability. Building on these findings, Martin Gellert and colleagues identified DNA gyrase in 1976 as the first Type II topoisomerase in bacteria. Using purified E. coli extracts and relaxed closed-circular DNA substrates, they observed ATP-dependent introduction of negative supercoils, monitored by shifts in sedimentation velocity from relaxed to supercoiled forms in neutral buffers. This enzyme, composed of GyrA and GyrB subunits, provided the counterforce to topoisomerase I's relaxing activity, establishing the enzymatic basis for maintaining physiological supercoiling levels in bacteria.

Key Milestones and Nomenclature

Following the initial discoveries of bacterial topoisomerases, the 1980s marked key advances in eukaryotic systems. Eukaryotic type I topoisomerase, first identified in mammalian cells in 1972 for its ability to relax supercoiled DNA, underwent detailed purification and characterization during this decade, confirming its ATP-independent activity in altering DNA topology. In 1980, eukaryotic type II topoisomerase was isolated and recognized for its critical role in mitosis, specifically through ATP-dependent decatenation of interlinked daughter chromosomes to enable proper segregation.^[12] The nomenclature for topoisomerases was established in the 1980s based on the number of DNA strands cleaved and energy requirements. Type I topoisomerases introduce transient single-strand breaks and are generally ATP-independent, facilitating relaxation of supercoils via either strand passage (type IA) or controlled rotation (type IB). Type II topoisomerases create double-strand breaks and depend on ATP hydrolysis to drive strand passage, enabling both relaxation and decatenation. Subtypes within these classes were defined by mechanistic distinctions: type IA enzymes, such as bacterial topoisomerase I, utilize an enzyme-bridged covalent intermediate for strand passage, while type IB enzymes, including eukaryotic topoisomerase I, permit controlled rotation of the DNA around the break site before religation.^[13] In the late 1980s and 1990s, further refinements expanded the classification. Type IC topoisomerases were introduced with the discovery of topoisomerase V from the hyperthermophilic archaeon Methanopyrus kandleri in 1993; these enzymes employ a tyrosyl-based phosphodiester bond similar to type IB but feature a unique five-domain structure and eukaryotic-like activity in extreme conditions. Type IIB topoisomerases emerged as a distinct group in archaea and some eukaryotes during this period, exemplified by the 1997 discovery of topoisomerase VI, which shares type II strand-passage capabilities but with specialized ATPase domains adapted for high-temperature environments.^[14] These developments built on pioneering contributions from researchers like James C. Wang, who coined the term "topoisomerase," and Leroy F. Liu, whose work on eukaryotic enzymes elucidated their roles in DNA metabolism and inspired targeted anticancer therapies.^[12]

Type I Topoisomerases

Type IA

Type IA topoisomerases constitute a subclass of type I enzymes that are ATP-independent and specialized for relaxing negative DNA supercoils through a strand passage mechanism. These enzymes are ubiquitous in bacteria, where they maintain DNA topology during essential processes like transcription; for instance, Escherichia coli DNA topoisomerase I (Topo I), encoded by the topA gene, selectively relaxes negative supercoils to counteract the action of DNA gyrase. They are also present in certain archaea, such as topoisomerase III in hyperthermophiles like Sulfolobus solfataricus, and in eukaryotes (e.g., topoisomerase III), though eukaryotic type IA enzymes like TOP3 primarily function in decatenation and recombination rather than supercoil relaxation.^[15]^[16]^[3] Structurally, type IA topoisomerases exhibit a five-domain architecture that assembles into a toroidal clamp capable of encircling single-stranded DNA. In bacterial examples like E. coli Topo I, domains I–III and V form the core catalytic region, while domain IV contributes to the clamp's stability and DNA binding. This configuration creates a protein hole approximately 20 Å in diameter, sufficient to accommodate a single DNA strand. Catalytic activity depends on Mg²⁺ ions, which coordinate with conserved aspartate residues in the active site to facilitate nucleophilic attack by a tyrosine residue on the DNA phosphodiester backbone.^[17]^[13]^[18] The mechanism employs a controlled strand passage model, wherein the enzyme covalently cleaves one DNA strand to form a gate, allowing an intact single strand to pass through before religation, thereby altering the linking number without rotational unwinding. This ATP-independent process relies on the toroidal structure to bridge and guide the DNA segments, enabling efficient relaxation of negative supercoils in a step-wise manner. Unlike type IB enzymes, type IA topoisomerases require single-stranded DNA regions for activity and operate unidirectionally on negatively supercoiled substrates.^[15]^[16]^[19] Type IA topoisomerases are notably inhibited by preexisting single-strand breaks in DNA, as the enzyme can form a persistent covalent adduct at the nick, impeding religation and potentially stalling replication forks. In many bacteria, these enzymes are essential for viability; for example, topA null mutants in E. coli exhibit lethal hyper-negative supercoiling unless suppressed by secondary mutations in gyrase genes, underscoring their critical role in genomic stability.^[3]^[20]^[16]

Type IB

Type IB topoisomerases are a subclass of type I enzymes primarily found in eukaryotic organisms, where they play a crucial role in managing DNA topology by relaxing supercoils through single-strand breaks.^[21] These enzymes are characterized by their ability to relax both positive and negative supercoils without requiring ATP or divalent cations, distinguishing them from other topoisomerase subtypes.^[22] In humans, the nuclear form, TOP1, and the mitochondrial form, TOP1MT, represent key examples, with TOP1 distributed throughout eukaryotic nuclei and TOP1MT localized to mitochondria in vertebrates.^[22] Additionally, type IB enzymes occur in certain viruses, such as poxviruses like vaccinia virus, which encodes a homolog sharing mechanistic similarities with eukaryotic versions.^[21] Structurally, type IB topoisomerases consist of a core domain and a C-terminal domain connected by a linker region of two extended α-helices.^[21] The core domain, exceeding 90 kDa in size, adopts a C-shaped clamp that non-covalently encircles duplex DNA, while the C-terminal domain houses the catalytic tyrosine residue—such as Tyr723 in human TOP1—that forms a transient 3'-phosphotyrosyl covalent intermediate with the cleaved DNA strand.^[21] Unlike type IA enzymes, type IB topoisomerases lack a clamp mechanism for strand passage; instead, they facilitate DNA relaxation through controlled rotation, where the free DNA end swivels around the intact strand in a friction-limited, stepwise manner.^[21] This swivel mechanism enables high processivity, with human TOP1 capable of up to 6000 cleavage-religation cycles per minute, allowing efficient resolution of torsional stress without enzyme dissociation.^[23] A distinctive property of type IB topoisomerases is their sensitivity to inhibition by camptothecin, a plant-derived alkaloid that binds at the enzyme-DNA interface, stacking against the +1 base and forming hydrogen bonds to stabilize the cleavage complex, thereby preventing religation and inducing DNA damage.^[23] These enzymes are essential for cellular viability, as TOP1 depletion leads to embryonic lethality in mice and impairs vertebrate cell proliferation due to unresolved supercoiling during transcription and replication.^[24] Similarly, TOP1MT supports mitochondrial genome stability, and its knockout results in increased negative supercoiling of mtDNA and defective liver regeneration.^[24] Mutations in TOP1MT, such as R111W, have been linked to spinocerebellar ataxia, causing bioenergetic deficits, mitochondrial DNA damage, and neurological symptoms in affected individuals.^[25]

Type IC

Type IC topoisomerases form a rare and structurally unique subfamily of type I enzymes, primarily exemplified by topoisomerase V (Topo V) from the hyperthermophilic archaeon Methanopyrus kandleri. This enzyme was discovered in 1993 through purification from M. kandleri extracts, marking the first identification of a prokaryotic type I topoisomerase with mechanistic similarities to eukaryotic type IB enzymes, though its classification as type IC emerged later based on its distinct features.^[14]41862-X/fulltext) Topo V represents an evolutionary outlier, with phylogenetic studies proposing it as a bridge between type I and other topoisomerase lineages due to its modular architecture combining catalytic and DNA-binding elements reminiscent of diverse enzyme families.^[26]^[27] The structure of Topo V diverges significantly from both type IA and type IB enzymes, featuring a novel α-helical fold in its ~30 kDa N-terminal topoisomerase domain that lacks homology to other known topoisomerases.^[28]^[29] Despite this, it shares the type IB-like mechanism of forming a reversible tyrosyl-DNA phosphodiester covalent intermediate via nucleophilic attack by a conserved active-site tyrosine (Tyr-226), which facilitates single-strand breakage and religation.^[30] The enzyme's overall ~110 kDa size includes a C-terminal extension with 12 tandem helix-hairpin-helix (HhH) motifs that mediate high-affinity DNA binding and contribute to thermostability, though truncated fragments as small as 61 kDa retain core catalytic activity.^[31] Gate dynamics differ from type IB enzymes, involving a constrained, stepwise rotation of DNA strands stabilized by salt bridges and hydrogen bonds, rather than uncontrolled swivel, which enables precise control under extreme conditions.^[32] Recent cryo-EM and crystal structures of Topo V-DNA complexes confirm these dynamics, revealing an unusual open conformation that accommodates DNA entry and exit through multiple subdomains.^[33] Type IC topoisomerases are distributed exclusively in select archaea, such as M. kandleri and possibly related hyperthermophiles, with no homologs identified in bacteria or most eukaryotes.^[21] Their restricted presence underscores an archaeal-specific adaptation, potentially acquired via horizontal gene transfer from an ancient virosphere, as suggested by comparative genomics indicating viral-like sequence divergence.^[27]^[26] These enzymes relax both negatively and positively supercoiled DNA via a rotation (swiveling) mechanism, allowing controlled rotation of the DNA around the break to relieve supercoils without net strand passage, at a rate of approximately 20 turns per second at 40°C under low torsional stress conditions.^[32] This activity supports DNA unwinding during replication and transcription in high-temperature environments, with Topo V's additional AP lyase function—catalyzed by three distinct active sites in its HhH domains—enabling cleavage at apurinic/apyrimidinic sites for repair, thus integrating topological and damage-response roles.^[31] In viral contexts, while no direct type IC enzymes occur in modern viruses, the enzyme's proposed viral ancestry implies an ancestral contribution to giant virus replication machinery, akin to type IB topoisomerases in nucleocytoviruses.^[27]

Type II Topoisomerases

Type IIA

Type IIA topoisomerases are a subclass of type II enzymes that function as homodimers in eukaryotes or heterotetramers (A₂B₂) in bacteria, featuring a conserved core architecture with distinct domains that enable DNA manipulation. The enzyme consists of an N-terminal ATPase domain responsible for ATP binding and hydrolysis, a B-gate (or TOPRIM) domain involved in DNA cleavage, and a C-terminal exit gate domain that facilitates strand passage. These domains assemble into a heart-shaped clamp structure, approximately 20 nm in size, which wraps around DNA segments to position them for processing, with three key gating interfaces: the N-gate for segment capture, the DNA-gate for cleavage and passage, and the C-gate for segment release.^[34]^[35] In bacteria, Type IIA topoisomerases are represented by DNA gyrase and topoisomerase IV (Topo IV), both essential for genome maintenance. DNA gyrase, composed of GyrA and GyrB subunits, actively introduces negative supercoils into DNA to counteract torsional stress during replication and transcription, wrapping about 130 base pairs of DNA in a right-handed manner to direct the topology. Topo IV, formed by ParC and ParE subunits, primarily decatenates interlinked daughter chromosomes after replication, showing a preference for positive supercoils and operating with high efficiency in disentangling. These enzymes are ubiquitous in bacterial species, with gyrase being vital for viability in most, such as Escherichia coli.^[36]^[35] Eukaryotes express two isoforms of Type IIA topoisomerases: topoisomerase IIα (TOP2A) and topoisomerase IIβ (TOP2B), both homodimeric proteins encoded by separate genes and differing in tissue distribution and regulation. TOP2A is highly expressed in proliferating cells and plays a critical role in mitosis by resolving catenanes and ensuring proper chromosome segregation, with peak activity during S and G2/M phases. In contrast, TOP2B is more abundant in differentiated and non-proliferating tissues, contributing to developmental processes and maintaining chromatin structure. Unlike bacterial counterparts, eukaryotic Type IIA enzymes do not introduce supercoils but relax both positive and negative supercoils in an ATP-dependent manner.^[36]^[37] A defining property of Type IIA topoisomerases is their ATP-dependent mechanism for double-strand DNA passage, where ATP binding triggers conformational changes to capture and transport a DNA segment through a transient break in another segment, preventing topological entanglements. This process is powered by hydrolysis of one or two ATP molecules per cycle, ensuring directionality and efficiency in vivo. Notably, bacterial DNA gyrase uniquely harnesses the free energy from ATP hydrolysis to drive the energetically unfavorable introduction of negative supercoils, a capability absent in other Type IIA enzymes, which instead use ATP primarily for relaxation or decatenation.^[35]^[38]

Type IIB

Type IIB topoisomerases constitute a distinct subfamily within the type II enzymes, characterized by their unique structural and functional adaptations for DNA manipulation. The prototypical member is DNA topoisomerase VI (topo VI), first identified and reconstituted from the hyperthermophilic archaeon Sulfolobus shibatae. These enzymes form heterotetramers composed of two catalytic A subunits and two ATPase-containing B subunits, differing from the homodimeric organization of many type IIA enzymes. The A subunit features a core catalytic domain responsible for generating double-strand breaks via a tyrosine residue that forms a covalent 5'-phosphotyrosyl linkage with DNA, along with a winged-helix domain that aids in DNA binding. The B subunit includes an N-terminal ATPase domain with limited sequence homology to type IIA ATPases, which dimerizes upon nucleotide binding to drive conformational changes. In eukaryotes, the type IIB homolog Spo11 functions in meiotic recombination by generating DNA double-strand breaks without full strand passage and religation.^[39]^[40]^[41]^[42] Structurally, type IIB topoisomerases exhibit a simplified gating system compared to type IIA counterparts, employing a two-gate mechanism for strand passage. The enzyme captures a T-segment (transported DNA) in a protein clamp formed by the ATPase domains (N-gate), followed by cleavage of a G-segment (gate DNA) at the DNA gate. Unlike type IIA enzymes, which possess an additional C-gate (exit gate) for DNA release, type IIB enzymes lack this feature; instead, the transported DNA is released through the open DNA gate after passage, relying on ATP hydrolysis to reset the enzyme. Crystal structures of the topo VI B subunit from Methanococcus jannaschii, solved in 2003, revealed the nucleotide-bound dimeric state and supported this model, while later intact enzyme structures from 2008 generalized the mechanism across type II families.^[43]^[44] High-resolution insights into gate mechanics have been further illuminated by cryo-EM studies in the 2010s, particularly for related Spo11 complexes in eukaryotes, confirming the conserved yet adapted gate dynamics in archaeal topo VI.^[45] Type IIB topoisomerases are predominantly distributed in archaea, where they play essential roles in DNA processing, with homologs also present in plants, algae, and certain eukaryotic organelles such as chloroplasts, but absent from most bacteria. In archaea like Sulfolobus species, topo VI efficiently decatenates linked DNA molecules without introducing or relaxing supercoils, showing a strong preference for unlinking crossings at near-right angles (approximately 87°), which confers chiral selectivity for positive writhe. This activity is ATP-dependent, with strand passage rates enhanced by DNA topology but occurring at lower efficiencies (up to ~6 events per minute) than type IIA enzymes. A notable function in archaea involves regulating plasmid copy number by facilitating decatenation during replication, ensuring proper segregation and stable maintenance of extrachromosomal elements.^[46]^[4]^[47]

Mechanisms of Action

Type I Mechanisms

Type I topoisomerases catalyze the relaxation of supercoiled DNA through a two-step mechanism involving strand breakage and religation, without requiring ATP hydrolysis; instead, the energy is derived from the phosphodiester bonds of the DNA backbone.^[21] The process begins with a nucleophilic attack by a conserved tyrosine residue in the enzyme's active site on the DNA phosphodiester bond, forming a transient covalent intermediate where the tyrosine is linked to the 3' phosphate (in type IB enzymes) or 5' phosphate (in type IA and IC enzymes) of the cleaved strand via transesterification.^[48]^[49] This covalent tyrosyl-DNA linkage anchors the broken strand to the enzyme, preventing dissociation and allowing controlled manipulation of the DNA topology.^[13] Following cleavage, the mechanism diverges between subtypes to achieve strand passage or rotation. In type IB enzymes, such as human topoisomerase IB, the non-covalently bound DNA segment rotates freely around the intact strand, allowing the unwinding of hundreds of superhelical turns in a controlled manner before religation; this rotation is facilitated by the enzyme's toroidal structure that clamps the DNA ends post-cleavage.^[48] In contrast, type IA and IC enzymes, like bacterial topoisomerase I and archaeal topoisomerase V, employ an enzyme-bridged strand passage mechanism, where the cleaved DNA strand passes through a short gate formed by the enzyme, typically in discrete steps of 2-3 base pairs, to alter topology incrementally without extensive free rotation.^[49]^[16] The rate of supercoil relaxation (k) depends on factors such as Mg²⁺ concentration and initial DNA topology; for type IA enzymes, Mg²⁺ is essential for strand passage and religation, with optimal rates at 2-4 mM, while supercoiled substrates are relaxed faster than relaxed DNA due to preferential binding.^[50]^[51] Type IB enzymes show less stringent Mg²⁺ dependence for cleavage but may require it for efficient religation in some contexts.^[50] Religation proceeds via the reverse transesterification reaction, where the 5'-hydroxyl (or 3'-hydroxyl in type IA/IC) of the cleaved strand attacks the phosphotyrosyl bond, reforming the DNA backbone and releasing the enzyme with high fidelity and minimal errors.^[48]^[49] This error-free process ensures genome integrity, as the covalent intermediate precisely aligns the DNA ends for accurate rejoining.^[13]

Type II Mechanisms

Type II topoisomerases mediate the passage of one double-stranded DNA segment (T-segment) through a transient double-strand break in another segment (G-segment), requiring ATP hydrolysis to drive the process and altering DNA topology by steps of two in linking number (ΔLk).^[3] The enzyme operates as a dimeric clamp with three key gates: the N-gate (ATPase domains), B-gate (cleavage site), and C-gate (exit portal), ensuring controlled strand passage.^[52] Fidelity is maintained through covalent phosphotyrosine intermediates formed during cleavage, which covalently link the enzyme to the DNA ends and prevent uncontrolled dissociation.^[53] The catalytic cycle begins with the G-segment binding across the B-gate, where the enzyme cleaves both strands with a 4-base stagger (in type IIA enzymes), creating 5'-phosphotyrosyl bonds while the 3'-OH ends remain free.^[3] ATP binding to the N-terminal ATPase domains induces dimerization and closure of the N-gate, capturing a nearby T-segment within the enzyme cavity.^[54] Hydrolysis of one ATP molecule per cycle opens the B-gate, allowing the T-segment to pass through the cleaved G-segment toward the C-gate.^[54] The T-segment then transits through the C-gate to exit, followed by religation of the G-segment via nucleophilic attack of the 3'-OH on the phosphotyrosine bond, restoring the DNA backbone and resetting the enzyme for another cycle.^[3] The process is powered by ATP hydrolysis, with a standard free energy change of ΔG ≈ -30 kJ/mol, providing directionality and preventing futile cycling by favoring forward strand passage. For DNA gyrase, each cycle introduces negative supercoils, changing the linking number by ΔLk = -2, as the wrapping of DNA around the enzyme (approximately 140 base pairs) couples passage to supercoil pumping.^[3] In contrast to gyrase, topoisomerase IV and type IIB enzymes like topoisomerase VI primarily facilitate decatenation rather than supercoiling, lacking the wrapping domain and focusing on resolving interlinked DNA molecules during segregation; type IIB enzymes produce a 2-base stagger upon cleavage.^[3]

Biological Roles

In DNA Replication and Repair

Topoisomerases play essential roles in DNA replication by alleviating the topological constraints that arise as the replication fork progresses. During replication, the unwinding of the double helix by helicases generates positive supercoils ahead of the fork, creating torsional stress that impedes fork advancement if not resolved. Both type IA and type IB topoisomerases relieve this stress: type IA enzymes, such as bacterial topoisomerase III, preferentially relax negatively supercoiled DNA but can also address positive supercoils in certain contexts, while type IB topoisomerases, like eukaryotic topoisomerase I, efficiently relax both positive and negative supercoils through single-strand breaks. Type II topoisomerases, including type IIA enzymes such as DNA gyrase in bacteria and topoisomerase II in eukaryotes, further contribute by introducing negative supercoils to counteract the positive torsion, ensuring smooth progression of the replisome. In bacteria, DNA gyrase actively maintains the appropriate superhelical density required for efficient replication initiation and elongation. At the termination of replication, type II topoisomerases, particularly topoisomerase IV in bacteria and topoisomerase II in eukaryotes, decatenate the interlinked daughter DNA molecules, separating the newly synthesized chromosomes to allow their segregation. Failure to decatenate leads to incomplete replication and genomic instability, as observed in topoisomerase II-deficient yeast cells where daughter molecules remain entangled.^[55]^[56]^[57]^[58]^[59]^[60] In DNA repair, topoisomerases resolve topological barriers that emerge during the repair of double-strand breaks and other lesions. In homologous recombination (HR), the invasion of a single-stranded DNA overhang into a homologous duplex generates extensive positive supercoiling behind the D-loop structure, which type IB topoisomerases like topoisomerase I relieve to facilitate branch migration and Holliday junction resolution. Type II topoisomerases also participate in HR by decatenating recombination intermediates, preventing their accumulation as toxic structures. For non-homologous end joining (NHEJ), topoisomerase II aids in the repair of topoisomerase-induced breaks by processing ends and facilitating ligation, particularly when covalent topoisomerase-DNA adducts trap the enzyme on damaged sites; defects in NHEJ pathways hypersensitize cells to topoisomerase II poisons, underscoring this role. These activities ensure that repair processes do not inadvertently introduce further topological strain. Additionally, topoisomerases resolve R-loops formed during replication stress, preventing transcription-replication conflicts and genomic instability.^[1]^[61]^[62]^[63]^[22] Illustrative examples highlight the critical nature of topoisomerases in replication and repair. In bacteria, inhibitors of DNA gyrase, such as quinolones like ciprofloxacin, stabilize the enzyme-DNA cleavage complex, preventing supercoil relaxation and halting replication fork progression, which leads to cell death. In eukaryotes, mutations in topoisomerase I, such as those impairing its cleavage-religation cycle, result in persistent DNA breaks and increased genomic instability, as the unresolved torsional stress promotes fork stalling and collapse. A key quantitative insight is that replication fork progression in eukaryotes is limited by topoisomerase activity, proceeding at rates of approximately 20-40 base pairs per second in systems like yeast and mammalian cells, where insufficient relaxation slows the helicase and polymerase.^[64]^[65]^[66]^[67]^[68]^[69]

In Transcription

During transcription, the advancing RNA polymerase unwinds DNA, generating positive supercoils ahead of the transcription fork and negative supercoils behind it, which can impede polymerase progression if unresolved. Topoisomerase I (TOP1) primarily relaxes these positive supercoils ahead of the polymerase by forming transient single-strand breaks, allowing strand rotation to relieve torsional stress and support efficient elongation.^[70] This activity is coupled to RNA polymerase II (RNAPII) pause-release, where phosphorylation of RNAPII's C-terminal domain enhances TOP1 processivity approximately 1.5 kb downstream of transcription start sites.^[70] In contrast, topoisomerase II (TOP2) resolves negative supercoils behind the polymerase and aids in promoter clearance by decatenating interlinked DNA segments that arise during initiation.^[71] In eukaryotic systems, topoisomerase IIβ (TOP2B), an isoform of TOP2, is particularly important for transcription initiation of developmentally regulated and stimulus-responsive genes. TOP2B binds preferentially to promoter regions, where it removes catenanes to facilitate open complex formation and RNAPII promoter escape.^[72] For instance, TOP2B regulates the immediate early gene c-fos by inducing targeted double-strand breaks at its promoter upon neuronal stimulation, such as via NMDA receptor activation, which promotes RNAPII pause-release and rapid gene induction.^[72]^[73] This mechanism ensures topological relief for enhancer-promoter looping and efficient transcription of long genes. TOP1 also plays a key role in the elongation phase, especially for genes with extended transcripts, by continuously mitigating supercoiling to prevent polymerase stalling.^[74] In TOP1-deficient models, such as shRNA knockdown in B cells, unresolved supercoils lead to increased RNAPII accumulation and enhanced transcription pausing, resulting in elevated polymerase density at transcribed loci.^[75] Recent research has further connected TOP1 activity to epigenetic maintenance in stress-prone genomic regions, such as those harboring stress-inducible genes; here, TOP1 relieves transcription-induced tension to enable nucleosome reassembly post-elongation, synergizing with chromatin remodelers like SWI/SNF to preserve chromatin structure and gene regulation under stress.^[76]

In Chromosome Segregation and Mitosis

During mitosis in eukaryotic cells, topoisomerase II (Topo II) plays a crucial role in resolving DNA catenations between sister chromatids, enabling their proper segregation in anaphase. Specifically, Topo IIα, the predominant isoform in proliferating cells, localizes to centromeres and chromosome arms, where it decatenates intertwined DNA strands generated during DNA replication, preventing entanglements that could lead to chromosome breakage or missegregation.^[22] This activity is tightly regulated and peaks during anaphase onset, coinciding with cohesin cleavage by separase, which allows the physical separation of chromatids pulled by the mitotic spindle. In parallel, topoisomerase I (Topo I) supports centromere function by facilitating transcription of centromeric satellite DNA, which aids in kinetochore assembly and overall chromosome stability during segregation.^[77] In eukaryotes, Topo IIα expression is cell cycle-regulated, with protein levels increasing through S phase and reaching a peak in G2/M to support chromosome condensation and segregation.^[78] Inhibition of Topo II activity, such as through catalytic blockade, disrupts this process, leading to persistent catenations, metaphase arrest, and ultimately aneuploidy due to unequal chromosome distribution.^[79] A unique regulatory mechanism involves sumoylation of Topo IIα, which modifies its localization and enzymatic activity at centromeres, fine-tuning mitotic timing and ensuring timely decatenation; defects in this post-translational modification impair chromosome segregation and are associated with genomic instability in cancer.^[80] In bacteria, the equivalent function is performed by topoisomerase IV (Topo IV), a type II enzyme that primarily acts post-replication to decatenate daughter chromosomes, allowing their segregation into daughter cells before division. Unlike eukaryotic Topo II, which handles both relaxation and decatenation throughout the cell cycle, Topo IV is specialized for disentangling replicated DNA and is recruited behind the replication fork, ensuring efficient chromosome partitioning in prokaryotes.^[81]

As Drug Targets

Antibacterial Inhibitors

Bacterial topoisomerases, particularly DNA gyrase and topoisomerase IV, are essential enzymes in prokaryotes that manage DNA supercoiling and decatenation, making them prime targets for antibacterial agents. Inhibitors of these enzymes disrupt DNA replication and repair, leading to bacterial cell death without significantly affecting eukaryotic topoisomerases due to structural differences. Fluoroquinolones, such as ciprofloxacin, represent the most clinically successful class of topoisomerase inhibitors, primarily targeting type IIA enzymes in bacteria. These synthetic compounds stabilize the cleavage complex formed by DNA gyrase or topoisomerase IV, preventing the religation of DNA strands and causing double-strand breaks that trigger cell death. Their mechanism involves binding to a magnesium ion (Mg²⁺)-chelated DNA-enzyme complex at the interface of the DNA gate, which inhibits the strand passage step in the catalytic cycle. Ciprofloxacin, for instance, exhibits broad-spectrum activity against Gram-negative and Gram-positive bacteria by dually inhibiting gyrase (primary target in Gram-negatives) and topoisomerase IV (primary in Gram-positives).^[82]^[83]^[82] Resistance to fluoroquinolones often arises through mutations in the quinolone resistance-determining regions (QRDRs) of the genes encoding these enzymes, such as gyrA and parC, which reduce drug affinity for the target complex. These point mutations, commonly in the GyrA and ParC subunits, alter the enzyme's DNA-binding interface and are a major driver of clinical resistance in pathogens like Escherichia coli and Staphylococcus aureus.^[82]^[84] Aminocoumarins, exemplified by novobiocin, offer an alternative mechanism by non-competitively inhibiting the ATPase activity of the GyrB subunit in DNA gyrase, thereby blocking the energy-dependent supercoiling of DNA. Novobiocin binds to the ATP-binding site on GyrB, preventing ATP hydrolysis and halting the enzyme's conformational changes required for DNA strand passage. This class has been used historically for treating Gram-positive infections, though limited by poor pharmacokinetics; novobiocin shows potent inhibition of gyrase with IC₅₀ values in the low micromolar range.^[85]^[86]^[87] Proteinaceous inhibitors like microcin B17, a ribosomally synthesized and post-translationally modified peptide produced by certain E. coli strains, act as a gyrase poison by covalently trapping the enzyme in a cleaved DNA complex. Microcin B17 enters target cells via specific transporters and inhibits gyrase after the DNA cleavage step, stabilizing the complex and inducing double-strand breaks similar to fluoroquinolones but without requiring metal ions. This leads to SOS response activation and bacterial lethality, with activity primarily against Enterobacteriaceae.^[88]^[89]^[90] Recent efforts to revive aminocoumarins have focused on novobiocin analogs optimized for improved solubility and reduced toxicity, with preclinical studies in 2023 demonstrating enhanced antibacterial potency against multidrug-resistant Neisseria gonorrhoeae through targeted modifications to the coumarin core. These analogs maintain GyrB inhibition while addressing historical limitations, positioning them as candidates for further development against resistant Gram-negative pathogens.^[91] Gepotidacin (Blujepa), a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor (NBTI), was approved by the FDA on March 25, 2025, for the treatment of uncomplicated urinary tract infections in female adults and adolescents (aged 12 years and older, weighing at least 40 kg) caused by susceptible isolates of Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis. It inhibits both DNA gyrase and topoisomerase IV by stabilizing the cleavage complex, offering a new class of antibiotics with low resistance potential. As of November 2025, it is under priority review for uncomplicated urogenital gonorrhea.^[92]^[93]

Anticancer Inhibitors

Topoisomerase inhibitors have become cornerstone agents in anticancer chemotherapy by exploiting the elevated DNA replication and transcription demands of rapidly proliferating tumor cells, which rely heavily on these enzymes for topological control. Classical inhibitors primarily target eukaryotic topoisomerase I (Topo I) and topoisomerase II (Topo II), stabilizing cleavage complexes to induce lethal DNA damage, particularly during S-phase or G2/M phases of the cell cycle. These small-molecule drugs, including derivatives of camptothecin and podophyllotoxin, have demonstrated efficacy across various solid tumors and hematologic malignancies, though their use is tempered by dose-limiting toxicities and emerging resistance mechanisms. Camptothecin derivatives, such as irinotecan, function as Topo I poisons by binding at the enzyme-DNA interface to trap the reversible cleavage complex, preventing religation and leading to persistent single-strand breaks that convert to double-strand breaks upon collision with replication forks. This mechanism causes S-phase-specific cell cycle arrest and apoptosis, particularly effective in colorectal, lung, and ovarian cancers. Irinotecan, a prodrug converted to the active metabolite SN-38, is commonly administered in regimens like FOLFIRI, with dosing adjusted based on UGT1A1 genotype to mitigate severe diarrhea; recent clinical insights from 2020s trials support variable doses of 120–350 mg/m² every two weeks, allowing higher tolerated levels in patients with favorable pharmacogenetics. Topotecan, another derivative, follows a similar trapping mechanism but is preferred for gynecologic malignancies due to its intravenous formulation and dosing of 1.25 mg/m² daily for five days. Etoposide, a semisynthetic podophyllotoxin derivative, acts as a Topo II poison by stabilizing the enzyme's DNA cleavage complex, inhibiting religation and generating double-strand breaks that trigger G2/M arrest and mitotic catastrophe, making it a standard component in regimens for small cell lung cancer, lymphomas, and germ cell tumors. Administered at 50–100 mg/m² intravenously over 30–60 minutes daily for three to five days, etoposide's efficacy stems from its selectivity for Topo IIα, the isoform overexpressed in proliferating cells, though prolonged exposure can exacerbate myelosuppression. In combination with platinum agents, it achieves response rates exceeding 60% in extensive-stage small cell lung cancer, with 2020s studies confirming stable dosing without major revisions but emphasizing supportive care to manage neutropenia. Doxorubicin, an anthracycline antibiotic, exerts its Topo II inhibitory effects through DNA intercalation, which enhances cleavage complex stabilization and blocks religation, resulting in DNA damage and apoptosis across breast, sarcoma, and leukemia treatments. Unlike pure poisons, its planar structure inserts between base pairs, amplifying cytotoxicity but also contributing to off-target effects; cumulative doses are capped at 400–550 mg/m² to limit cardiotoxicity, which arises from Topo IIβ-mediated oxidative stress and mitochondrial dysfunction in cardiomyocytes. Recent 2020s analyses underscore mitigation strategies like dexrazoxane chelation, allowing safer escalation in high-risk patients while preserving antitumor activity. Merbarone, a thiobarbituric acid derivative, represents a less common non-intercalating catalytic inhibitor of Topo II, binding the enzyme to prevent ATP-dependent DNA strand passage without stabilizing cleavage complexes, thereby inducing minimal DNA damage and reduced mutagenesis compared to poisons. Evaluated in phase II trials for renal cell carcinoma and colorectal cancer in the 1990s, it showed modest response rates (10–20%) at doses of 400–1200 mg/m² daily for five days but has seen limited recent adoption due to variable efficacy and hypersensitivity risks. Its unique profile highlights potential for combination therapies avoiding excessive genotoxicity. A key challenge in utilizing these inhibitors is acquired resistance, often mediated by efflux pumps such as ABCB1 (P-glycoprotein) and ABCC1, which actively export drugs from tumor cells, reducing intracellular concentrations and diminishing therapeutic efficacy in relapsed settings. Multidrug resistance phenotypes, prevalent in up to 50% of refractory cases, underscore the need for pharmacogenomic screening and adjunctive modulators, though clinical translation remains limited.

Emerging Therapeutic Strategies

Antibody-drug conjugates (ADCs) represent a significant advancement in topoisomerase-targeted therapies by enabling precise delivery of topoisomerase I (topo I) inhibitors to cancer cells expressing specific surface antigens, thereby minimizing systemic toxicity associated with traditional chemotherapeutics. Enhertu (fam-trastuzumab deruxtecan), which conjugates a HER2-directed monoclonal antibody with the topo I inhibitor DXd, received initial FDA approval in 2019 for HER2-positive metastatic breast cancer and has since expanded to include HER2-mutant non-small cell lung cancer (NSCLC) in 2022, unresectable or metastatic HER2-positive solid tumors in April 2024, and hormone receptor-positive, HER2-low or ultra-low breast cancer in January 2025. Similarly, Trodelvy (sacituzumab govitecan), linking a Trop-2-directed antibody to the topo I inhibitor SN-38, was first approved in 2020 for metastatic triple-negative breast cancer, followed by hormone receptor-positive, HER2-negative breast cancer in February 2023 and breakthrough therapy designation for second-line extensive-stage small cell lung cancer in December 2024. Datroway (datopotamab deruxtecan), another TROP2-directed ADC with a topo I inhibitor payload (deruxtecan), was approved in January 2025 for previously treated metastatic hormone receptor-positive, HER2-negative breast cancer and in June 2025 for previously treated locally advanced or metastatic EGFR-mutated NSCLC. These expansions have broadened access for patients with breast and lung cancers, leveraging targeted payload release upon antigen binding and lysosomal cleavage to induce DNA damage selectively in tumor cells.^[94]^[95]^[96]^[97]^[98]^[99]^[100] Recent developments in novel topo I and topo II inhibitors focus on dual-targeting agents to enhance efficacy against resistant tumors, with several candidates advancing in clinical trials. Dual topo I/II inhibitors, such as derivatives inspired by pixantrone—a topo II poison approved for non-Hodgkin lymphoma—aim to simultaneously disrupt both enzyme activities, potentially overcoming compensatory mechanisms in cancer cells; preclinical analogs of pixantrone have shown promise in modulating topo IIα inhibition while exploring topo I synergy, though no dual agents are yet approved. Complementing this, epigenetic modulation of topo I has emerged as a strategy to exploit tumor-specific vulnerabilities, particularly in regions of topological stress. A 2025 study in Nature Communications demonstrated that epigenetic regulators control topo I localization and activity in stress-prone genomic loci, suggesting that inhibitors targeting these modifiers could sensitize cancers to topo I poisons by altering chromatin accessibility and enzyme recruitment.^[101]^[102]^[103] Combination therapies pairing topo I inhibitors with other DNA repair modulators are showing synergistic potential, particularly in colorectal cancer. A 2024 study highlighted the synergy between topo I inhibitors like topotecan and human DNA ligase I (hLig I) inhibitors, where co-inhibition traps DNA breaks and impairs repair, leading to enhanced cytotoxicity in colorectal cancer cells without excessive toxicity to normal tissues; these findings build on 2023 preclinical data demonstrating reduced tumor growth in models resistant to monotherapy. Such approaches address resistance by exploiting synthetic lethality in repair pathways. The global market for topo I inhibitors is projected to grow from $5.7 billion in 2024 to $8.1 billion by 2031, driven by ADC expansions and combination regimens that suppress resistance through multi-pathway targeting.^[104]^[105]^[106]^[107]

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Dec 17, 2024 · U.S. FDA Grants Breakthrough Therapy Designation to Trodelvy sacituzumab govitecan hziy for Second Line Treatment of Extensive Stage Small ...Missing: expansions 2023
[94]
New Life of Topoisomerase I Inhibitors as Antibody Drug Conjugate ...
Two ADC delivering topoisomerase I (TOP1) poisons (Enhertu and Trodelvy) have recently been FDA-approved for Her2- and Trop2-expressing solid tumors.
[95]
Topoisomerase II-targeting anticancer clinical candidates and drugs
Jul 5, 2025 · We present a structure-function analysis of human topoisomerase II (hTopo II) inhibitors those are currently clinically used or under clinical trials for ...
[96]
Multiple Topoisomerase I (TopoI), Topoisomerase II (TopoII) and ...
To date, no dual topoisomerase inhibitor is clinically approved, which is either due to low/missing antitumor activity or clinically relevant toxicity (11)(12)( ...
[97]
Epigenetic control of topoisomerase 1 activity presents a cancer ...
Aug 12, 2025 · Unresolved TOP1ccs have been linked to somatic mutations, chromosomal aberrations and cell death. Consistent with this, genetic defects in ...
[98]
Synergy between human DNA ligase I and topoisomerase 1 unveils ...
Jan 5, 2024 · Our findings offer evidence for the synergistic impact of combining hLig I inhibitors with Topotecan in the treatment of colorectal cancers.Missing: 2023 | Show results with:2023
[99]
Synergy between human DNA ligase I and topoisomerase 1 unveils ...
Jan 5, 2024 · In conclusion, our findings offer evidence for the synergistic impact of combining hLig I inhibitors with Topotecan in the treatment of ...
[100]
Recent developments of topoisomerase inhibitors: Clinical trials ...
In this review, we provide an overview of the clinical drug development highlighting advances in current clinical uses and discussing novel drugs and ...
[101]
Global Topoisomerase I Inhibitors Sales Market Report, Competitive ...
The global Topoisomerase I Inhibitors market size was US$ 5726 million in 2024 and is forecast to a readjusted size of US$ 8111 million by 2031 with a CAGR ...Missing: projection | Show results with:projection