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The many lives of type IA topoisomerases

  • Anna H. Bizard
    Correspondence
    To whom correspondence may be addressed. Tel: 45-35335482;
    Affiliations
    Center for Chromosome Stability and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark
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  • Ian D. Hickson
    Correspondence
    To whom correspondence may be addressed. Tel.: 45-35326738;
    Affiliations
    Center for Chromosome Stability and Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark
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Open AccessPublished:April 10, 2020DOI:https://doi.org/10.1074/jbc.REV120.008286
      The double-helical structure of genomic DNA is both elegant and functional in that it serves both to protect vulnerable DNA bases and to facilitate DNA replication and compaction. However, these design advantages come at the cost of having to evolve and maintain a cellular machinery that can manipulate a long polymeric molecule that readily becomes topologically entangled whenever it has to be opened for translation, replication, or repair. If such a machinery fails to eliminate detrimental topological entanglements, utilization of the information stored in the DNA double helix is compromised. As a consequence, the use of B-form DNA as the carrier of genetic information must have co-evolved with a means to manipulate its complex topology. This duty is performed by DNA topoisomerases, which therefore are, unsurprisingly, ubiquitous in all kingdoms of life. In this review, we focus on how DNA topoisomerases catalyze their impressive range of DNA-conjuring tricks, with a particular emphasis on DNA topoisomerase III (TOP3). Once thought to be the most unremarkable of topoisomerases, the many lives of these type IA topoisomerases are now being progressively revealed. This research interest is driven by a realization that their substrate versatility and their ability to engage in intimate collaborations with translocases and other DNA-processing enzymes are far more extensive and impressive than was thought hitherto. This, coupled with the recent associations of TOP3s with developmental and neurological pathologies in humans, is clearly making us reconsider their undeserved reputation as being unexceptional enzymes.

      Introduction

      In B-form DNA, the two complementary strands are associated with one another via hydrogen bonds that form between the bases of each strand. These paired strands periodically coil around their longitudinal axis in a clockwise orientation, giving rise to a so-called right-handed double helix. The intertwining that results from this conformation prevents the separation of the strands and thereby ensures that the genetic information is tightly protected within the center of the DNA fiber. However, to gain access to the sequences of the bases, the double helix must be opened, which requires the topological disentanglement of the two strands. In that respect, although topological intertwines provide stability to the double helix, they nevertheless can be viewed as a fundamental obstacle to the functionality of DNA. Hence, organisms must strictly control the degree to which their genomic DNA is intertwined to guarantee both the protection and the functionality of their genetic material.
      B-form DNA is widely recognized for its elegant double-helical structure. However, this double helix is not as static as it is generally represented. It is instead constantly manipulated to extract, duplicate, and repair the genetic information that it encodes. The reciprocal interplay between DNA topology and DNA metabolism has an overwhelming complexity because many, if not all, factors involved in these processes alter the topological entanglements of the genome. Among these factors, the machineries that translocate along DNA clearly play one of the most prominent roles. When a translocating protein machinery is not free to rotate around the axis of the DNA, it instead behaves as a topological barrier whose translocation drives the rotation of the DNA fiber itself. In a topologically constrained DNA molecule, this DNA rotation leads to the overwinding (positive supercoiling) of the DNA segment located ahead of the translocase, whereas the DNA in its wake becomes progressively underwound (negatively supercoiled) (Fig. 1A). This process describes the so-called “twin supercoiling domain” model in which the absolute degree of interstrand entanglement of the DNA molecule is not altered, but instead is distributed differently relative to the position of the translocase (
      • Liu L.F.
      • Wang J.C.
      Supercoiling of the DNA template during transcription.
      ). In living cells, the twin supercoiling domain model primarily applies to the translocation of the transcription machinery, whose rotation is thought to be limited not only by its size, but also by steric constraints, such that RNA polymerases introduce substantial levels of both positive and negative supercoiling (
      • Ma J.
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      DNA supercoiling during transcription.
      ,
      • Kouzine F.
      • Gupta A.
      • Baranello L.
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      • Ben-Aissa K.
      • Liu J.
      • Przytycka T.M.
      • Levens D.
      Transcription-dependent dynamic supercoiling is a short-range genomic force.
      ,
      • Teves S.S.
      • Henikoff S.
      Transcription-generated torsional stress destabilizes nucleosomes.
      ,
      • Naughton C.
      • Avlonitis N.
      • Corless S.
      • Prendergast J.G.
      • Mati I.K.
      • Eijk P.P.
      • Cockroft S.L.
      • Bradley M.
      • Ylstra B.
      • Gilbert N.
      Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures.
      ,
      • Giaever G.N.
      • Wang J.C.
      Supercoiling of intracellular DNA can occur in eukaryotic cells.
      ) (Fig. 1B). This superhelical constraint has been shown to impact many DNA processes, including DNA replication, chromatin and chromosome architecture, and transcription itself (
      • Teves S.S.
      • Henikoff S.
      Transcription-generated torsional stress destabilizes nucleosomes.
      ,
      • Naughton C.
      • Avlonitis N.
      • Corless S.
      • Prendergast J.G.
      • Mati I.K.
      • Eijk P.P.
      • Cockroft S.L.
      • Bradley M.
      • Ylstra B.
      • Gilbert N.
      Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures.
      ,
      • Roca J.
      Transcriptional inhibition by DNA torsional stress.
      ). Indeed, because they represent an excess of topological linkages, the positive supercoils generated ahead of any RNA polymerase prevent DNA opening required for transcription initiation and would ultimately block the progression of the polymerase if not dealt with (
      • Baranello L.
      • Wojtowicz D.
      • Cui K.
      • Devaiah B.N.
      • Chung H.J.
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      • Wilson K.
      • Zhang X.
      • Zhang H.
      • Piotrowski J.
      • Thomas C.J.
      • Singer D.S.
      • Pugh B.F.
      • Pommier Y.
      • Przytycka T.M.
      • Kouzine F.
      • Lewis B.A.
      • Zhao K.
      • Levens D.
      RNA polymerase II regulates topoisomerase 1 activity to favor efficient transcription.
      ,
      • Gartenberg M.R.
      • Wang J.C.
      Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast.
      ,
      • Joshi R.S.
      • Piña B.
      • Roca J.
      Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes.
      ,
      • Ma J.
      • Bai L.
      • Wang M.D.
      Transcription under torsion.
      ). Conversely, the negative supercoils generated behind the transcription machinery correspond to a deficit in topological linkages and can lead to the destabilization of the double helix, an effect that favors the formation of R-loops and secondary DNA structures, which threaten the continued stability of the genome (
      • Sollier J.
      • Cimprich K.A.
      Breaking bad: R-Loops and genome integrity.
      ,
      • Skourti-Stathaki K.
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      A double-edged sword: R loops as threats to genome integrity and powerful regulators of gene expression.
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      R Loops: From Physiological to Pathological Roles.
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      • Stolz R.
      • Sulthana S.
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      • Chedin F.
      Interplay between DNA sequence and negative superhelicity drives R-loop structures.
      ).
      Figure thumbnail gr1
      Figure 1Topological constraints associated with DNA metabolism in vivo. A, twin supercoiling domain model. When a translocating machinery is not allowed to rotate around the DNA axis (arrowhead with black circle), it introduces overwinding (positive supercoiling; +ve SC) in front of and underwinding (negative supercoiling; −ve SC) behind the translocase. B, in the context of transcription, the overwinding accumulated ahead of the RNA polymerase prevents strand opening and can ultimately block transcription elongation. Underwinding generated behind the polymerase can promote strand opening and lead to the stabilization of R-loops and other secondary structures. C, during DNA replication, and when fork rotation is prevented, the degree of entanglement between the newly replicated DNA molecules is limited, but overwinding ahead of the fork can prevent replisome progression. D, during DNA replication, overwinding of the template can be limited by fork rotation, but this leads to formation of precatenanes behind the fork, which represent an obstacle during segregation.
      The twin supercoiling domain model also applies to the translocation of the DNA replication machinery (replisome), although the potential topological problems arising during this process are more complicated due to the additional presence of the two sister dsDNA molecules that form behind the replication fork (
      • Keszthelyi A.
      • Minchell N.E.
      • Baxter J.
      The causes and consequences of topological stress during DNA replication.
      ,
      • Schvartzman J.B.
      • Stasiak A.
      A topological view of the replicon.
      ). Most often, it would seem that the rotation of the replisome is prevented, such that DNA synthesis is associated with the generation of positive supercoils in the unreplicated template DNA (
      • Le T.T.
      • Gao X.
      • Park S.H.
      • Lee J.
      • Inman J.T.
      • Lee J.H.
      • Killian J.L.
      • Badman R.P.
      • Berger J.M.
      • Wang M.D.
      Synergistic coordination of chromatin torsional mechanics and topoisomerase activity.
      ) (Fig. 1C). If allowed to accumulate, these supercoils would prevent further progression of the replisome and promote fork stalling and potentially fork reversal (
      • Postow L.
      • Ullsperger C.
      • Keller R.W.
      • Bustamante C.
      • Vologodskii A.V.
      • Cozzarelli N.R.
      Positive torsional strain causes the formation of a four-way junction at replication forks.
      ). The deficit in topological entanglements behind the translocating replisome manifests as a limited degree of winding of each of the newly replicated sister chromatids. However, under special circumstances, such as during replication termination or at sites of replication-transcription conflicts, the build-up of positive supercoils has been proposed to trigger fork rotation (
      • Dewar J.M.
      • Budzowska M.
      • Walter J.C.
      The mechanism of DNA replication termination in vertebrates.
      ,
      • Sundin O.
      • Varshavsky A.
      Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers.
      ,
      • Schalbetter S.A.
      • Mansoubi S.
      • Chambers A.L.
      • Downs J.A.
      • Baxter J.
      Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability.
      ) (Fig. 1D). In such cases, the intramolecular topological linkages between the template strands are converted into intertwines between the newly replicated sister DNA segments (
      • Champoux J.J.
      • Been M.D.
      Topoisomerases and the swivel problem.
      ). These intertwines (known as precatenanes) would be converted into catenanes upon completion of replication and are generally considered to be the main obstacle to the faithful segregation of the sister chromatids during cell division.
      Given the topological challenges that are continually generated during DNA processes such as transcription and replication, it is inconceivable that B-form DNA could have evolved as a carrier of genetic information without the co-evolution of efficient ways to actively relieve topological stress (
      • Watson J.D.
      • Crick F.H.C.
      Genetical implications of the structure of deoxyribonucleic acid.
      ). This is particularly critical for circular genomes and for long linear chromosomes whose organization is effectively constrained topologically because their structure does not allow the torsional energy to be dissipated by free rotations of their ends (
      • Naughton C.
      • Avlonitis N.
      • Corless S.
      • Prendergast J.G.
      • Mati I.K.
      • Eijk P.P.
      • Cockroft S.L.
      • Bradley M.
      • Ylstra B.
      • Gilbert N.
      Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures.
      ,
      • Joshi R.S.
      • Piña B.
      • Roca J.
      Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes.
      ,
      • Dixon J.R.
      • Selvaraj S.
      • Yue F.
      • Kim A.
      • Li Y.
      • Shen Y.
      • Hu M.
      • Liu J.S.
      • Ren B.
      Topological domains in mammalian genomes identified by analysis of chromatin interactions.
      ). By promoting DNA breakage, strand rotation, and ligation within a three-step catalytic cycle, DNA topoisomerases are the enzymes responsible for manipulating the absolute degree of topological entanglement of DNA (
      • Champoux J.J.
      DNA topoisomerases: structure, function, and mechanism.
      ). They achieve this prodigious feat by catalyzing a reversible transesterification reaction involving an active-site tyrosine residue and a phospho-ester bond of the DNA substrate. This reaction leads to the formation of the so-called topoisomerase cleavage complex, a topoisomerase-DNA intermediate in which the enzyme is covalently attached to an extremity of the broken DNA. This bond can be reversed by another transesterification reaction, enabling the nick to be resealed and the enzyme to be released from its covalent association with the substrate. Using this elegant biochemical trick, DNA topoisomerases are able to manipulate intra- and intermolecular DNA topology without exposing their substrates to the dangers of long-lived strand breaks.
      DNA topoisomerases belong to a select group of enzymes that are conserved in all living organisms and that existed during the earliest stages of cellular life. All bacteria, archaea, and eukaryotic cells are equipped with a set of DNA topoisomerases that is sufficient to cope with the topological by-products of their DNA metabolism by releasing negative and positive supercoils, as well as intermolecular catenation. In eukaryotic cells, these activities are performed by TOP1 and TOP2, which are members of the Type IB and Type IIA families of topoisomerases, respectively (Fig. 2). TOP1 introduces a transient nick into one strand of dsDNA, which leads to the formation of a covalent linkage to the 3′ end of the nick (Fig. 2A). Efficient release of both negative and positive supercoils is then performed via a swiveling mechanism during which the 5′ end of the scissile strand rotates freely around the intact DNA strand to dissipate torsional stress and reset the molecule to its most stable topological conformation (
      • Kim N.
      • Jinks-Robertson S.
      The Top1 paradox: friend and foe of the eukaryotic genome.
      ) (Fig. 2B). Type II topoisomerases can also relax both positive and negative supercoils, but they do so via an intrinsically different mechanism, which involves active manipulation of the DNA entanglements by the enzyme. Eukaryotic TOP2s are homodimeric enzymes that catalyze transesterification reactions on both strands of a dsDNA substrate, leading to the formation of a cleavage complex in which each protomer is covalently associated with a 5′ end of a double-stranded break (Fig. 2C). The 3′ extremities of this break, which are not covalently bound to the catalytic tyrosine residues, are nevertheless tightly associated with the enzyme and thereby prevented from rotating. Instead, changes in topology occur because an intact dsDNA molecule is actively transported through the transient dsDNA break (
      • Champoux J.J.
      DNA topoisomerases: structure, function, and mechanism.
      ). This mode of action is intrinsically distributive and potentially riskier than a swiveling mechanism for DNA relaxation, but it enables Type II topoisomerases to modulate both intra- and intermolecular DNA topology, the latter being critical for the decatenation of sister chromatids formed during DNA replication (Fig. 2D).
      Figure thumbnail gr2
      Figure 2Mechanism of action and catalytic activities of Type IB and Type II topoisomerases. A, i, Type IB topoisomerases bind to dsDNA. ii, a transesterification reaction leads to the formation of a single strand break with the 3′ terminus covalently associated with the active tyrosine. Torsional stress in the substrate is dissipated by free rotation of the 5′ end of the nick. iii, a second transesterification reaction religates the nick and frees the enzyme from its covalent interaction. Open red circle, catalytic tyrosine; closed red circle, catalytic tyrosine engaged in a covalent DNA intermediate. B, Type IB topoisomerases efficiently relax both positive (+ve SC) and negative (−ve SC) supercoils. C, i, Type II topoisomerases are dimeric enzymes that possess two catalytic tyrosine residues (open red circles). ii, transesterification reactions lead to the introduction of a transient double strand break into a dsDNA molecule (G-segment; purple). After cleavage, each 5′ end of the DNA break is covalently associated with one of the tyrosines (closed red circles). Conformational changes in the protein brings the ends of the broken G-segment apart, enabling another dsDNA molecule (T-segment; yellow) to pass though the gate. iii, a second set of transesterification reactions religate the break and free the enzyme from its covalent interaction with the DNA. D, when the G-segments (purple) and T-segments (yellow) are located on the same molecule, Type II topoisomerase activity leads to the relaxation of negative and positive supercoils (top). When the G- and T-segments are located in trans, Type II topoisomerases can modify the degree of catenation between two dsDNA molecules (bottom).
      The different biochemical activities of eukaryotic Type IB and Type II topoisomerases have evolved to conduct at least some distinct functions in vivo. Whereas Type IB topoisomerases are well-suited to roles in relieving torsional stress generated during transcription and replication, Type II topoisomerases are indispensable for the decatenation of DNA that is necessary during chromosome condensation and segregation. The well-characterized relaxation and decatenation activities of Type IB and Type II topoisomerases appear sufficient to manage all topological constraints associated with DNA metabolism in eukaryotes (
      • Brill S.J.
      • DiNardo S.
      • Voelkel-Meiman K
      • Sternglanz R.
      Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA.
      ,
      • Bermejo R.
      • Doksani Y.
      • Capra T.
      • Katou Y.M.
      • Tanaka H.
      • Shirahige K.
      • Foiani M.
      Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation.
      ,
      • Baxter J.
      • Diffley J.F.X.
      Topoisomerase II inactivation prevents the completion of DNA replication in budding yeast.
      ,
      • Pommier Y.
      • Sun Y.
      • Huang S.N.
      • Nitiss J.L.
      Roles of eukaryotic topoisomerases in transcription, replication and genomic stability.
      ,
      • Lee J.H.
      • Berger J.M.
      Cell cycle-dependent control and roles of DNA topoisomerase II.
      ,
      • Austin C.A.
      • Lee K.C.
      • Swan R.L.
      • Khazeem M.M.
      • Manville C.M.
      • Cridland P.
      • Treumann A.
      • Porter A.
      • Morris N.J.
      • Cowell I.G.
      TOP2B: the first thirty years.
      ). It is perhaps surprising, therefore, that every eukaryotic cell also expresses another class of topoisomerase, the Type IA enzymes (
      • Garnier F.
      • Debat H.
      • Nadal M.
      Type IA DNA topoisomerases: a universal core and multiple activities.
      ,
      • Viard T.
      • de la Tour C.B.
      Type IA topoisomerases: a simple puzzle?.
      ,
      • Bugreev D.V.
      • Nevinsky G.A.
      Structure and mechanism of action of type IA DNA topoisomerases.
      ). Indeed, Type IA topoisomerases are the only topoisomerases conserved in virtually all living organisms. They are further divided into three subfamilies, the TOP3 enzymes, bacterial TOPA enzymes, and reverse gyrases. Whereas the distribution of TOPAs and reverse gyrases is restricted to bacteria and hyperthermophilic organisms (
      • Forterre P.
      • Gribaldo S.
      • Gadelle D.
      • Serre M.C.
      Origin and evolution of DNA topoisomerases.
      ), most organisms encode for at least one TOP3. Although it is commonly accepted that TOP3s are absolutely critical for the maintenance of genomic stability, their precise cellular function has remained quite mysterious. This is probably because they essentially lack the canonical relaxation activity shared by all other DNA topoisomerases and hence have not been considered important for the release of torsional stress generated by transcription and replication. These Type IA topoisomerases can nevertheless manipulate a broad range of topological substrates in vitro. The physiological relevance of this catalytic versatility is now being progressively uncovered, which has defined the involvement of TOP3s in some unexpected cellular processes. In this review, we will focus on our current knowledge of this unique family of topoisomerases and the many roles that they play in the maintenance of the stability of our genomes.

      TOP3 topoisomerase biochemistry

      Protein structure and catalytic cycle

      All Type IA topoisomerases share a very similar architecture within their core topoisomerase domain, which comprises one topoisomerase-primase subdomain (TOPRIM;
      The abbreviations used are: TOPRIM
      topoisomerase-primase subdomain
      CAP
      catabolite activator protein subdomain
      T-segment
      transported segment
      UFB
      ultrafine anaphase bridge
      cen-UFBs
      ultrafine anaphase bridges that arise from centromeres
      mtDNA
      mitochondrial DNA
      RMI
      RecQ-mediated genome instability
      OB-fold
      oligonucleotide-oligosaccharide–binding fold
      ssDNA and ssRNA
      single-stranded DNA and RNA, respectively.
      subdomain I) and two catabolite activator protein subdomains (CAP; subdomains III and IV), which are connected by two topo-folds (subdomain II) (
      • Goto-Ito S.
      • Yamagata A.
      • Takahashi T.S.
      • Sato Y.
      • Fukai S.
      Structural basis of the interaction between Topoisomerase IIIβ and the TDRD3 auxiliary factor.
      ,
      • Mondragón A.
      • DiGate R.
      The structure of Escherichia coli DNA topoisomerase III.
      ,
      • Hansen G.
      • Harrenga A.
      • Wieland B.
      • Schomburg D.
      • Reinemer P.
      Crystal structure of full length topoisomerase I from Thermotoga maritima.
      ,
      • Bocquet N.
      • Bizard A.H.
      • Abdulrahman W.
      • Larsen N.B.
      • Faty M.
      • Cavadini S.
      • Bunker R.D.
      • Kowalczykowski S.C.
      • Cejka P.
      • Hickson I.D.
      • Thomä N.H.
      Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1.
      ,
      • Lima C.D.
      • Wang J.C.
      • Mondragón A.
      Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I.
      ,
      • Rodríguez A.C.
      • Stock D.
      Crystal structure of reverse gyrase: Insights into the positive supercoiling of DNA.
      ,
      • Capranico G.
      • Marinello J.
      • Chillemi G.
      Type I DNA topoisomerases.
      ) (Fig. 3A). Overall, this core domain resembles a toroidal clamp in which subdomains I, III, and IV are associated at the base of an arc formed by subdomain II. The clamp can adopt either a closed or open conformation upon association or dissociation of a gate formed at the interface between subdomain III and subdomains I and IV. Besides the catalytic tyrosine, which is located within subdomain III, the transesterification reaction requires 10 highly conserved residues distributed throughout subdomains I, III, and IV (
      • Garnier F.
      • Debat H.
      • Nadal M.
      Type IA DNA topoisomerases: a universal core and multiple activities.
      ). As a result, the catalytic pocket can only be constituted at the interface of the gate upon closure of the clamp (Fig. 3A).
      Figure thumbnail gr3
      Figure 3Structure and catalytic cycle of Type IA topoisomerases. A, the structure of the core topoisomerase domain resembles a toroidal clamp in which the topo-fold subdomain II forms an arc. The catalytic site is reconstituted at the base of this arc, by the association of residues from the topoisomerase-primase (TOPRIM) subdomain I and the two catabolite activator (CAP-Y and CAP) subdomains III and IV. The deep ssDNA (and ssRNA) binding groove (G-segment–binding groove) is formed between subdomains I and IV. B, a, Type IA topoisomerases bind to single-stranded segments of DNA via the G-segment–binding groove that directs the G-segment in line with the catalytic tyrosine (i, open red circle). b, a transesterification reaction leads to the formation of a single strand break with the 5′ termini covalently associated with the catalytic tyrosine (ii, closed red circle). The 3′ end of the nick forms a tight association with the G-segment–binding groove. c, protein conformational changes enable the opening of a gate via the separation of the CAP domains and their associated DNA termini (iii). d, another nucleic acid segment (here the single-strand DNA complementary to the G-segment) is passed though the gate toward the cavity of the enzyme (iv). e, after this transport, the closure of the gate reconstitutes the catalytic cycle (v). f, a second transesterification reaction reseals the nick and frees the enzyme from its covalent interaction with the substrate (vi). g, full dissociation is enabled by opening of the gate.
      Although many aspects of the mechanism by which Type IA topoisomerases manipulate DNA topology remain to be understood, a model for their mode of action has been proposed (
      • Bugreev D.V.
      • Nevinsky G.A.
      Structure and mechanism of action of type IA DNA topoisomerases.
      ,
      • Dekker N.H.
      • Rybenkov V.V.
      • Duguet M.
      • Crisona N.J.
      • Cozzarelli N.R.
      • Bensimon D.
      • Croquette V.
      The mechanism of type IA topoisomerases.
      ) (Fig. 3B). In this model, the enzyme associates with its substrate via a DNA-binding groove primarily created by subdomains I and IV, which guides the scissile DNA strand (or gated segment; G-segment) toward the catalytic pocket, such that it is in line with the active tyrosine of the CAP subdomain III (
      • Changela A.
      • DiGate R.J.
      • Mondragón A.
      Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule.
      ,
      • Changela A.
      • DiGate R.J.
      • Mondragón A.
      Structural studies of E. coli topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA conformational intermediate.
      ). After G-segment nicking, Type IA topoisomerases and their substrates are engaged in a covalent interaction between the catalytic tyrosine of subdomain III and the terminal 5′-phosphate of the nick. A series of structural rearrangements involving the flexible subdomain II lead to a swinging motion of subdomain III relative to the subdomains I/IV, which opens the gate and drives the subdomains III and I/IV and their associated DNA termini apart (
      • Feinberg H.
      • Lima C.D.
      • Mondragón A.
      Conformational changes in E. coli DNA topoisomerase I.
      ,
      • Mills M.
      • Tse-Dinh Y.C.
      • Neuman K.C.
      Direct observation of topoisomerase IA gate dynamics.
      ,
      • Li Z.
      • Mondragón A.
      • DiGate R.J.
      The mechanism of type IA topoisomerase-mediated DNA topological transformations.
      ,
      • Xiong B.
      • Burk D.L.
      • Shen J.
      • Luo X.
      • Liu H.
      • Shen J.
      • Berghuis A.M.
      The type IA topoisomerase catalytic cycle: a normal mode analysis and molecular dynamics simulation.
      ). In this conformation, the noncovalent association between the enzyme and the 3′ terminus of the G-segment can still resist forces up to 40 picoNewtons, indicating that the enzyme and the 3′ terminus must remain very tightly associated (
      • Mills M.
      • Tse-Dinh Y.C.
      • Neuman K.C.
      Direct observation of topoisomerase IA gate dynamics.
      ,
      • Sarlós K.
      • Biebricher A.S.
      • Bizard A.H.
      • Bakx J.A.M.
      • Ferreté-Bonastre A.G.
      • Modesti M.
      • Paramasivam M.
      • Yao Q.
      • Peterman E.J.G.
      • Wuite G.J.L.
      • Hickson I.D.
      Reconstitution of anaphase DNA bridge recognition and disjunction.
      ,
      • Zhang Z.
      • Cheng B.
      • Tse-Dinh Y.C.
      Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase.
      ). This strong interaction, which is most likely mediated via the DNA-binding groove, also prevents the swiveling of the noncovalently attached DNA end. Instead, changes in DNA topology occur via the active transport of another nucleic acid molecule through the gate (
      • Dekker N.H.
      • Rybenkov V.V.
      • Duguet M.
      • Crisona N.J.
      • Cozzarelli N.R.
      • Bensimon D.
      • Croquette V.
      The mechanism of type IA topoisomerases.
      ). Once the transported segment (T-segment) has been passed through the gate, closure of the clamp leads to the reconstitution of the catalytic cycle. This in turn enables a second transesterification reaction to restore the continuity of the scissile strand and to free the enzyme and its substrate from their covalent interaction. Full dissociation is achieved by a final opening of the gate, allowing the T-segment to exit the cavity of the enzyme.

      Catalytic versatility

      Type IA topoisomerases are able to act on a broad range of topological structures, which contrasts with the limited number of substrates identified for other topoisomerase families (Fig. 4). Indeed, Type IA topoisomerases require only that their substrate exhibits a single-stranded segment to be used as a G-segment (
      • Kim R.A.
      • Wang J.C.
      Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase.
      ). This substrate restriction is determined by the nature of the G-segment–binding groove, whose dimensions can only accommodate a single-stranded nucleic acid segment with the bases facing toward the groove (
      • Mondragón A.
      • DiGate R.
      The structure of Escherichia coli DNA topoisomerase III.
      ,
      • Changela A.
      • DiGate R.J.
      • Mondragón A.
      Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule.
      ,
      • Changela A.
      • DiGate R.J.
      • Mondragón A.
      Structural studies of E. coli topoisomerase III-DNA complexes reveal a novel type IA topoisomerase-DNA conformational intermediate.
      ). This requirement explains why Type IA topoisomerases can relax supercoils only when the density of negative supercoiling is sufficiently high to promote strand separation, whereas they are much less potent on moderately negative and positive supercoils (unless a stable unpaired segment has been introduced into the substrate) (
      • Dekker N.H.
      • Rybenkov V.V.
      • Duguet M.
      • Crisona N.J.
      • Cozzarelli N.R.
      • Bensimon D.
      • Croquette V.
      The mechanism of type IA topoisomerases.
      ,
      • Kim R.A.
      • Wang J.C.
      Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase.
      ,
      • Plank J.L.
      • Chu S.H.
      • Pohlhaus J.R.
      • Wilson-Sali T.
      • Hsieh T.S.
      Drosophila melanogaster topoisomerase IIIα preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.
      ,
      • Chen C.F.
      • Brill S.J.
      Binding and activation of DNA topoisomerase III by the Rmi1 subunit.
      ,
      • Kirkegaard K.
      • Wang J.C.
      Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop.
      ,
      • Srivenugopal K.S.
      • Lockshon D.
      • Morris D.R.
      Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme.
      ). Similarly, the activity of these topoisomerases is promoted by the presence of DNA secondary structures, such as mismatches, G-quadruplexes, D-loops, and R-loops, which generate ssDNA regions in the substrate (
      • Yang Y.
      • McBride K.M.
      • Hensley S.
      • Lu Y.
      • Chedin F.
      • Bedford M.T.
      Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.
      ,
      • Wilson-Sali T.
      • Hsieh T.S.
      Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIβ.
      ,
      • Temime-Smaali N.
      • Guittat L.
      • Sidibe A.
      • Shin-ya K.
      • Trentesaux C.
      • Riou J.F.
      The G-quadruplex ligand telomestatin impairs binding of topoisomerase IIIα to G-quadruplex-forming oligonucleotides and uncaps telomeres in ALT cells.
      ,
      • Fasching C.L.
      • Cejka P.
      • Kowalczykowski S.C.
      • Heyer W.D.
      Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.
      ). Hence, Type IA topoisomerases are often described as single strand–specific DNA topoisomerases (
      • Wilson-Sali T.
      • Hsieh T.S.
      Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIβ.
      ). In addition to catalyzing topological conversions in cis, which lead to substrate relaxation, they can perform topological conversions in trans and thereby display decatenation activities (Fig. 4). Furthermore, the nature of the T-segment also appears to be unrestricted by the properties of the conserved core topoisomerase domain. In particular, although the ability of Type IA topoisomerases to transfer dsDNA molecules has not been demonstrated formally, both the width of the opened gate and the size of the cavity (around 25 Å) are sufficient to accommodate dsDNA (
      • Mills M.
      • Tse-Dinh Y.C.
      • Neuman K.C.
      Direct observation of topoisomerase IA gate dynamics.
      ). Moreover, Escherichia coli Top3 has been shown to be able to entrap a dsDNA T-segment in its closed cavity (
      • Li Z.
      • Mondragón A.
      • DiGate R.J.
      The mechanism of type IA topoisomerase-mediated DNA topological transformations.
      ). Last, but not least, Type IA topoisomerases have been shown to accept RNA both as a G- and as a T-segment and are therefore able to manipulate the topology of RNAs in vitro (
      • Ahmad M.
      • Xu D.
      • Wang W.
      An assay for detecting RNA topoisomerase activity.
      ,
      • Wang H.
      • Di Gate R.J.
      • Seeman N.C.
      An RNA topoisomerase.
      ,
      • DiGate R.J.
      • Marians K.J.
      Escherichia coli topoisomerase III-catalyzed cleavage of RNA.
      ,
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Ahmad M.
      • Xu D.
      • Wang W.
      Type IA topoisomerases can be “magicians” for both DNA and RNA in all domains of life.
      ). Hence, depending on the exact nature of the T-segment, members of this family have been shown to mediate the resolution of the full range of DNA and RNA intermolecular entanglements, including dsDNA catenanes, ssDNA catenanes, precatenanes, and hemicatenanes as well as knotted and catenated RNAs (
      • Ahmad M.
      • Xu D.
      • Wang W.
      An assay for detecting RNA topoisomerase activity.
      ,
      • Wang H.
      • Di Gate R.J.
      • Seeman N.C.
      An RNA topoisomerase.
      ,
      • DiGate R.J.
      • Marians K.J.
      Escherichia coli topoisomerase III-catalyzed cleavage of RNA.
      ,
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Ahmad M.
      • Xu D.
      • Wang W.
      Type IA topoisomerases can be “magicians” for both DNA and RNA in all domains of life.
      ,
      • Lee S.H.
      • Siaw G.E.L.
      • Willcox S.
      • Griffith J.D.
      • Hsieh T.S.
      Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases.
      ,
      • Cejka P.
      • Plank J.L.
      • Dombrowski C.C.
      • Kowalczykowski S.C.
      Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.
      ,
      • Nurse P.
      • Levine C.
      • Hassing H.
      • Marians K.J.
      Topoisomerase III can serve as the cellular decatenase in Escherichia coli.
      ,
      • DiGate R.J.
      • Marians K.J.
      Identification of a potent decatenating enzyme from Escherichia coli.
      ,
      • Yang J.
      • Bachrati C.Z.
      • Hickson I.D.
      • Brown G.W.
      BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity.
      ,
      • Bizard A.H.
      • Yang X.
      • Débat H.
      • Fogg J.M.
      • Zechiedrich L.
      • Strick T.R.
      • Garnier F.
      • Nadal M.
      TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.
      ,
      • Yang J.
      • Bachrati C.Z.
      • Ou J.
      • Hickson I.D.
      • Brown G.W.
      Human topoisomerase IIIα is a single-stranded DNA decatenase that is stimulated by BLM and RMI1.
      ) (Fig. 4). Hence, the conserved core catalytic domain of Type IA topoisomerases is characterized by impressive versatility, being able to modulate the topology of a broad range of DNA and RNA substrates. However, it is worth mentioning that some individual members of the type IA DNA topoisomerases appear to have become specialized for a subset of these reactions. This is well-illustrated by the catalytic, structural, and functional divergences of the two eukaryotic TOP3s, TOP3A and TOP3B, and will be discussed further below.
      Figure thumbnail gr4
      Figure 4Range of substrates for Type IA topoisomerases as a function of the nature of the G- and T- segments.
      The precise mechanism by which the transfer of the T-segment through the gate occurs is unknown. Type IA topoisomerases can relax both negative and positive supercoils in substrates with unpaired DNA regions. This property indicates that they can both increase and decrease intramolecular DNA entanglements and suggests that the directionality of any topological change is driven primarily by the existing topology of the substrate (
      • Plank J.
      • Hsieh T.S.
      Helicase-appended topoisomerases: new insight into the mechanism of directional strand transfer.
      ). Similar to their Type II counterparts, Type IA topoisomerases have been proposed to modify DNA topology via a so-called sign inversion mechanism. An alternative hypothesis is that these topoisomerases differentially alter DNA topology by the direction in which they transfer the T-segment either into or out of the cavity. Consistent with this alternative model, ecTop3 can capture a dsDNA segment into its closed cavity in the absence of a G-segment, indicating that the enzyme may be able to initiate its catalytic cycle when a T-segment is already located inside the cavity (
      • Li Z.
      • Mondragón A.
      • DiGate R.J.
      The mechanism of type IA topoisomerase-mediated DNA topological transformations.
      ) (Fig. 3B).

      Synergy with other enzymes

      Because of the intrinsic lack of substrate specificity and strand transfer directionality, the activity of Type IA topoisomerases is prone to be influenced by any factor that can alter the dynamics of dsDNA denaturation (including changes in temperature, the presence of ssDNA-binding proteins, or the activities of other DNA-processing enzymes) (
      • DiGate R.J.
      • Marians K.J.
      Identification of a potent decatenating enzyme from Escherichia coli.
      ,
      • Yang J.
      • Bachrati C.Z.
      • Hickson I.D.
      • Brown G.W.
      BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity.
      ,
      • Bizard A.H.
      • Yang X.
      • Débat H.
      • Fogg J.M.
      • Zechiedrich L.
      • Strick T.R.
      • Garnier F.
      • Nadal M.
      TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.
      ,
      • Plank J.
      • Hsieh T.S.
      Helicase-appended topoisomerases: new insight into the mechanism of directional strand transfer.
      ,
      • Kim Y.C.
      • Lee J.
      • Koo H.S.
      Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIα.
      ,
      • Xue X.
      • Raynard S.
      • Busygina V.
      • Singh A.K.
      • Sung P.
      Role of replication protein a in double Holliday junction dissolution mediated by the BLM-Topo IIIα-RMI1-RMI2 protein complex.
      ,
      • Reckinger A.R.
      • Jeong K.S.
      • Khodursky A.B.
      • Hiasa H.
      RecA can stimulate the relaxation activity of topoisomerase I: molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli.
      ,
      • Chen S.H.
      • Chan N.-L.
      • Hsieh T.S.
      New mechanistic and functional insights into DNA topoisomerases.
      ,
      • Wu L.
      • Hickson I.D.
      The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIα.
      ). Most notably, TOP3s are known for their ability to tightly cooperate with DNA translocases to catalyze complex topological conversions that could not be performed by a stand-alone topoisomerase. A good example is the so-called “dissolvasome” that results from the association of a TOP3 and a RecQ helicase (Schizosaccharomyces pombe Rqh1, Saccharomyces cerevisiae Sgs1, and human BLM) (
      • Ahmad F.
      • Stewart E.
      The N-terminal region of the Schizosaccharomyces pombe RecQ helicase, Rqh1p, physically interacts with Topoisomerase III and is required for Rqh1p function.
      ,
      • Wu L.
      • Davies S.L.
      • North P.S.
      • Goulaouic H.
      • Riou J.F.
      • Turley H.
      • Gatter K.C.
      • Hickson I.D.
      The Bloom's syndrome gene product interacts with topoisomerase III.
      ,
      • Gangloff S.
      • McDonald J.P.
      • Bendixen C.
      • Arthur L.
      • Rothstein R.
      The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.
      ) (Fig. 5). In this multienzyme complex, the activities of the RecQ helicase and TOP3 coordinate with each other to catalyze the dissolution of double Holliday junctions, which proceeds by the migration of the junctions toward each other until they merge to form a hemicatenated structure that is processed by TOP3 (
      • Wu L.
      • Hickson I.D.
      The Bloom's syndrome helicase suppresses crossing over during homologous recombination.
      ). In addition to its role during the unlinking of this hemicatenane, TOP3 also assists the convergent branch migration reaction primarily driven by the ATP-dependent helicase activity of Rqh1/Sgs1/BLM (
      • Chen S.H.
      • Plank J.L.
      • Willcox S.
      • Griffith J.D.
      • Hsieh T.S.
      Top3α is required during the convergent migration step of double holliday junction dissolution.
      ,
      • Chen S.H.
      • Wu C.H.
      • Plank J.L.
      • Hsieh T.S.
      Essential functions of C terminus of Drosophila topoisomerase IIIα in double Holliday junction dissolution.
      ,
      • Plank J.L.
      • Wu J.
      • Hsieh T.S.
      Topoisomerase IIIα and Bloom's helicase can resolve a mobile double Holliday junction substrate through convergent branch migration.
      ). The mechanism by which TOP3 manipulates DNA topology during the convergent branch migration is still not clear, although it seems likely that the activity of the RecQ helicase specifically enables TOP3 to efficiently release the torsional stress associated with convergent branch migration (
      • Bizard A.H.
      • Hickson I.D.
      The dissolution of double Holliday junctions.
      ). Besides its ability to dissolve double Holliday junctions, the dissolvasome has also been shown to catalyze the resolution of other complex substrates, such as D-loops, late-replication intermediates, and catenated dsDNA (
      • Sarlós K.
      • Biebricher A.S.
      • Bizard A.H.
      • Bakx J.A.M.
      • Ferreté-Bonastre A.G.
      • Modesti M.
      • Paramasivam M.
      • Yao Q.
      • Peterman E.J.G.
      • Wuite G.J.L.
      • Hickson I.D.
      Reconstitution of anaphase DNA bridge recognition and disjunction.
      ,
      • Fasching C.L.
      • Cejka P.
      • Kowalczykowski S.C.
      • Heyer W.D.
      Top3-Rmi1 dissolve Rad51-mediated D loops by a topoisomerase-based mechanism.
      ,
      • Cejka P.
      • Plank J.L.
      • Dombrowski C.C.
      • Kowalczykowski S.C.
      Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.
      ,
      • Lee C.M.
      • Wang G.
      • Pertsinidis A.
      • Marians K.J.
      Topoisomerase III acts at the replication fork to remove precatenanes.
      ,
      • Suski C.
      • Marians K.J.
      Resolution of converging replication forks by RecQ and topoisomerase III.
      ,
      • Harmon F.G.
      • DiGate R.J.
      • Kowalczykowski S.C.
      RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination.
      ,
      • Harmon F.G.
      • Brockman J.P.
      • Kowalczykowski S.C.
      RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III.
      ) (Fig. 5B). Altogether, therefore, this helicase-topoisomerase combination must be viewed as a multistructure dissolvasome, specialized in the resolution of various types of complex intermolecular entanglements (
      • Mankouri H.W.
      • Hickson I.D.
      The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific “dissolvasome”?.
      ).
      Figure thumbnail gr5
      Figure 5Functions of the RecQ helicase/Type IA topoisomerase “dissolvasome”. A, the dissolvasome is a multienzyme complex that combines the helicase activity of a RecQ family member and the topoisomerase activity of a TOP3 topoisomerase. RecQ helicases act as ssDNA translocases that rotate around the DNA axis (arrowhead with open black circle), such that the unpaired strands remain topologically entangled. In a covalently closed DNA molecule, the full dissociation of two paired strands requires the rupture of the hydrogen bounds (catalyzed by a helicase) and the dissipation of the topological entanglements resulting from the double-helical structure of DNA (catalyzed by a topoisomerase). B, synergistic cooperation between the helicase and topoisomerase activities of the dissolvasome enables the resolution of complex intermolecular entanglements, such as double Holliday junctions, late replication intermediates, and dsDNA catenanes.
      More recently, the human topoisomerase IIIα, TOP3A, was shown to cooperate with an ATPase of the SNF2 family, the dsDNA translocase PICH, to catalyze the introduction of positive supercoils into DNA (
      • Bizard A.H.
      • Allemand J.F.
      • Hassenkam T.
      • Paramasivam M.
      • Sarlós K.
      • Singh M.I.
      • Hickson I.D.
      PICH and TOP3A cooperate to induce positive DNA supercoiling.
      ) (Fig. 6). This supercoiling reaction is driven by the ability of PICH to act as a translocating topological barrier and to redistribute torsional stress on a dsDNA substrate, in the same manner as discussed above in the context of the twin supercoiling domain model (Fig. 1A). Unexpectedly, PICH was shown to extrude DNA loops concomitant with this torsional stress redistribution, such that negative supercoils are able to accumulate in the extruded loop located in its wake, whereas the rest of the substrate becomes progressively more positively supercoiled (
      • Bizard A.H.
      • Allemand J.F.
      • Hassenkam T.
      • Paramasivam M.
      • Sarlós K.
      • Singh M.I.
      • Hickson I.D.
      PICH and TOP3A cooperate to induce positive DNA supercoiling.
      ). Such a loop extrusion mechanism enables PICH to promote the local melting of the DNA in the loop, which creates the necessary hypernegatively supercoiled substrate for TOP3A to catalyze DNA relaxation. The final result of this combined activity of PICH and TOP3A is the introduction of an impressively high density of positive supercoiling in a processive manner (Fig. 6).
      Figure thumbnail gr6
      Figure 6Positive supercoiling activity of the PICH-TOP3A complex. PICH is a dsDNA translocase that extrudes DNA loops. Because it is prevented from rotating around the DNA axis (arrowhead with black circle), its translocation is associated with the redistribution of DNA torsional stress. This leads to an accumulation of negative (−ve SC) and positive (+ve SC) supercoils within and outside of the extruded loop, respectively. TOP3 relaxes the highly negatively supercoiled loop, which leads to an accumulation of net positive supercoiling in the substrate.
      The multienzyme complex formed by PICH and TOP3A is not the first example of synergistic cooperation between a Type IA topoisomerase and an ATPase to induce positive supercoiling. Indeed, such an activity is the hallmark of the so-called reverse gyrases, an atypical family of Type IA topoisomerases that are essential for the survival of hyperthermophilic organisms at high temperatures (
      • Perugino G.
      • Valenti A.
      • D'amaro A.
      • Rossi M.
      • Ciaramella M.
      Reverse gyrase and genome stability in hyperthermophilic organisms.
      ,
      • Lulchev P.
      • Klostermeier D.
      Reverse gyrase: recent advances and current mechanistic understanding of positive DNA supercoiling.
      ). Reverse gyrases are chimeric enzymes that combine a Type IA topoisomerase domain and an ATPase domain of the superfamily 2 helicases within the same polypeptide, which is reminiscent of the association between the human TOP3A and PICH proteins (
      • Rodríguez A.C.
      • Stock D.
      Crystal structure of reverse gyrase: Insights into the positive supercoiling of DNA.
      ). In contrast to their human analog, the hyperthermophilic reverse gyrases are distributive, and the degree of positive supercoiling they introduce is quite modest, illustrating a fundamentally different mechanism of action. Indeed, the ATPase domain of reverse gyrase is believed to function as a nucleotide switch that facilitates local DNA melting via a helicase-like mechanism (
      • Lulchev P.
      • Klostermeier D.
      Reverse gyrase: recent advances and current mechanistic understanding of positive DNA supercoiling.
      ). However, it is worth mentioning that some hyperthermophilic reverse gyrases, as exemplified by Sulfolobus solfataricus TopR2, introduce a high density of positive supercoiling in a processive manner (
      • Bizard A.
      • Garnier F.
      • Nadal M.
      TopR2, the second reverse gyrase of sulfolobus solfataricus, exhibits unusual properties.
      ). These observations are consistent with the existence of two reverse gyrase subfamilies that possess fundamentally different mechanisms of action and may indicate that the TopR2 reverse gyrases introduce positive supercoiling via a loop extrusion mechanism analogous to that of PICH and TOP3A.

      The cellular functions of TOP3 enzymes

      For DNA topoisomerases, just as for any other DNA-manipulating enzyme, a clear definition of their biochemical activities is a prerequisite for understanding their roles in vivo. The large repertoire of activities catalyzed by TOP3s alone and within multienzymatic complexes supports the notion that these topoisomerases are pleiotropic, being involved in a broad range of cellular processes that require the manipulation of specific topological structures that cannot be resolved by Type IB and Type II topoisomerases. Although TOP3s have been well-characterized in vitro, our understanding of their physiological roles is relatively superficial. However, several studies, in particular of the eukaryotic isoforms of TOP3s, are progressively revealing multiple roles of these unique topoisomerases in many aspects of cellular metabolism associated with the maintenance of genomic stability. Most higher eukaryotes encode two isoforms of TOP3, topoisomerases IIIα and IIIβ, designated TOP3A and TOP3B, respectively (Fig. 7A). Phylogenetic analysis indicates that the presence of these two isoforms resulted from a duplication early in the eukaryotic lineage (
      • Forterre P.
      • Gribaldo S.
      • Gadelle D.
      • Serre M.C.
      Origin and evolution of DNA topoisomerases.
      ). In the next section, we will review the known physiological roles of the two eukaryotic TOP3 isoforms.
      Figure thumbnail gr7
      Figure 7Domain organization of Type IA topoisomerases and their obligatory subunits. A, all Type IA topoisomerases share a highly conserved catalytic domain (blue) and sometimes an additional C-terminal extension (CTD), which contains multiple zinc finger motifs (black boxes) involved in protein-DNA and protein-protein interactions. In addition to putative zinc finger motifs, the C-terminal domain of HsTOP3B also exhibits RGG box motifs (green) that can be methylated and mediate interactions with RNA and the Tudor domain of TDRD3. An alternative start codon leads to the addition of a mitochondrial targeting sequence (MTS) to the TOP3A polypeptide, such that TOP3A encodes both nuclear and mitochondrial isoforms. In eukaryotes, TOP3 forms heterodimers with members of the RMI family. RMI members are characterized by a conserved association between a DUF1767/OB-fold domain (DUF-OB). In humans, the nuclear isoforms of TOP3A and TOP3B interact with their own cognate RMI protein, RMI1 and TDRD3, respectively. The RMI1 CTD exhibits a second OB-fold domain and mediates interactions with other proteins, including RMI2. TDRD3 CTD is characterized by the presence of multiple protein-protein interaction motifs including a ubiquitin-associating domain (UBA) and a Tudor domain. The Tudor domain of TDRD3 mediates interactions with methylated proteins, including histones, RNA polymerase, and TOP3B, and with the fragile X mental retardation protein. Ec, E. coli; Sc, S. cerevisiae; Hs, Homo sapiens. B, RMI1 and TDRD3 interact with the arc of TOP3A and TOP3B, respectively. RMI1 inserts a loop into the cavity of TOP3A, which restricts its size. A similar insertion loop is present in TDRD3, but this does not appear to significantly reduce the size of the TOP3B cavity.

      Roles of TOP3A during homologous recombination

      The function of eukaryotic TOP3 during homologous recombination has long been associated with its role as a member of the dissolvasome alongside RecQ helicases. The fission yeast top3+ gene is essential for cell viability, and the lethal phenotype of top3 mutants is suppressed by deletion of the gene encoding the Rqh1 RecQ helicase (
      • Goodwin A.
      • Wang S.W.
      • Toda T.
      • Norbury C.
      • Hickson I.D.
      Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe.
      ). In budding yeast, top3 mutants show a slow-growth phenotype that is suppressed by SGS1 gene mutations (
      • Gangloff S.
      • McDonald J.P.
      • Bendixen C.
      • Arthur L.
      • Rothstein R.
      The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase.
      ,
      • Wallis J.W.
      • Chrebet G.
      • Brodsky G.
      • Rolfe M.
      • Rothstein R.
      A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase.
      ). Moreover, top3 (and sgs1) mutants are synthetically lethal when combined with strains lacking the Mus81-Mms4 structure-selective nuclease, and this lethality can be suppressed by eliminating homologous recombination, indicating that Top3 and Sgs1 play an important role during some aspect of this process (
      • Oakley T.J.
      • Goodwin A.
      • Chakraverty R.K.
      • Hickson I.D.
      Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants.
      ). Although the dissolvasome could prevent early recombination events by dissolving D-loops, it clearly also acts during the later stages of some homologous recombination events by catalyzing double Holliday junction dissolution (
      • Wu L.
      • Hickson I.D.
      The Bloom's syndrome helicase suppresses crossing over during homologous recombination.
      ,
      • Bachrati C.Z.
      • Borts R.H.
      • Hickson I.D.
      Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase.
      ). Through its activity as a dissolution enzyme, not only for double Holliday junctions but also various other substrates (Fig. 5B), the dissolvasome influences many aspects of genome maintenance, including DNA replication, DNA repair, telomere maintenance, and meiosis (
      • Tsai H.J.
      • Huang W.H.
      • Li T.K.
      • Tsai Y.L.
      • Wu K.J.
      • Tseng S.F.
      • Teng S.C.
      Involvement of topoisomerase III in telomere-telomere recombination.
      ,
      • Tang S.
      • Wu M.K.Y.
      • Zhang R.
      • Hunter N.
      Pervasive and essential roles of the top3-rmi1 decatenase orchestrate recombination and facilitate chromosome segregation in meiosis.
      ,
      • Kaur H.
      • De Muyt A.
      • Lichten M.
      Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates.
      ,
      • Wang T.F.
      • Kung W.M.
      Supercomplex formation between Mlh1-Mlh3 and Sgs1-Top3 heterocomplexes in meiotic yeast cells.
      ,
      • Gangloff S.
      • de Massy B.
      • Arthur L.
      • Rothstein R.
      • Fabre F.
      The essential role of yeast topoisomerase III in meiosis depends on recombination.
      ,
      • Fabre F.
      • Chan A.
      • Heyer W.D.
      • Gangloff S.
      Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication.
      ,
      • Kim R.A.
      • Caron P.R.
      • Wang J.C.
      Effects of yeast DNA topoisomerase III on telomere structure.
      ,
      • Huang P.
      • Pryde F.E.
      • Lester D.
      • Maddison R.L.
      • Borts R.H.
      • Hickson I.D.
      • Louis E.J.
      SGS1 is required for telomere elongation in the absence of telomerase.
      ). Of the two eukaryotic TOP3 isoforms, only TOP3A has strongly conserved homology with its yeast Top3 counterpart, whereas TOP3B appears to have diverged significantly (Fig. 7A). TOP3A has an essential function, and its inactivation leads to embryonic lethality in mice, Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana (
      • Plank J.L.
      • Chu S.H.
      • Pohlhaus J.R.
      • Wilson-Sali T.
      • Hsieh T.S.
      Drosophila melanogaster topoisomerase IIIα preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.
      ,
      • Kim Y.C.
      • Lee J.
      • Koo H.S.
      Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIα.
      ,
      • Li W.
      • Wang J.C.
      Mammalian DNA topoisomerase IIIα is essential in early embryogenesis.
      ). Human subjects carrying hypomorphic TOP3A mutations are characterized by short stature and microcephaly (
      • Martin C.A.
      • Sarlós K.
      • Logan C.V.
      • Thakur R.S.
      • Parry D.A.
      • Bizard A.H.
      • Leitch A.
      • Cleal L.
      • Ali N.S.
      • Al-Owain M.A.
      • Allen W.
      • Altmüller J.
      • Aza-Carmona M.
      • Barakat B.A.Y.
      • Barraza-García J.
      • et al.
      Mutations in TOP3A cause a Bloom syndrome-like disorder.
      ). Cells with defective TOP3A have increased levels of sister chromatid exchanges and mitotic abnormalities, highlighting an essential role of TOP3A in the maintenance of genome stability that is reminiscent of BLM deficiency (
      • Martin C.A.
      • Sarlós K.
      • Logan C.V.
      • Thakur R.S.
      • Parry D.A.
      • Bizard A.H.
      • Leitch A.
      • Cleal L.
      • Ali N.S.
      • Al-Owain M.A.
      • Allen W.
      • Altmüller J.
      • Aza-Carmona M.
      • Barakat B.A.Y.
      • Barraza-García J.
      • et al.
      Mutations in TOP3A cause a Bloom syndrome-like disorder.
      ,
      • Hemphill A.W.
      • Akkari Y.
      • Newell A.H.
      • Schultz R.A.
      • Grompe M.
      • North P.S.
      • Hickson I.D.
      • Jakobs P.M.
      • Rennie S.
      • Pauw D.
      • Hejna J.
      • Olson S.B.
      • Moses R.E.
      Topo IIIα and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents.
      ,
      • Seki M.
      • Nakagawa T.
      • Seki T.
      • Kato G.
      • Tada S.
      • Takahashi Y.
      • Yoshimura A.
      • Kobayashi T.
      • Aoki A.
      • Otsuki M.
      • Habermann F.A.
      • Tanabe H.
      • Ishii Y.
      • Enomoto T.
      Bloom helicase and DNA topoisomerase III are involved in the dissolution of sister chromatids.
      ,
      • Chan K.L.
      • North P.S.
      • Hickson I.D.
      BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges.
      ). Consistently, TOP3A has been shown to be the topoisomerase component of the human dissolvasome together with the BLM helicase. In agreement with yeast genetic studies, this eukaryotic dissolvasome appears to be involved in multiple aspects of DNA metabolism that require homologous recombination or analogous processes, such as recovery from replication stress, alternative lengthening of telomeres, and regulation of meiotic crossovers (
      • Knoll A.
      • Schröpfer S.
      • Puchta H.
      The RTR complex as caretaker of genome stability and its unique meiotic function in plants.
      ,
      • Hartung F.
      • Suer S.
      • Knoll A.
      • Wurz-Wildersinn R.
      • Puchta H.
      Topoisomerase 3α and RMI1 suppress somatic crossovers and are essential for resolution of meiotic recombination intermediates in Arabidopsis thaliana.
      ,
      • Yang J.
      • O'Donnell L.
      • Durocher D.
      • Brown G.W.
      RMI1 promotes DNA replication fork progression and recovery from replication fork stress.
      ,
      • Temime-Smaali N.
      • Guittat L.
      • Wenner T.
      • Bayart E.
      • Douarre C.
      • Gomez D.
      • Giraud-Panis M.J.
      • Londono-Vallejo A.
      • Gilson E.
      • Amor-Guéret M.
      • Riou J.F.
      Topoisomerase IIIα is required for normal proliferation and telomere stability in alternative lengthening of telomeres.
      ,
      • Manthei K.A.
      • Keck J.L.
      The BLM dissolvasome in DNA replication and repair.
      ,
      • Lu R.
      • O'Rourke J.J.
      • Sobinoff A.P.
      • Allen J.A.M.
      • Nelson C.B.
      • Tomlinson C.G.
      • Lee M.
      • Reddel R.R.
      • Deans A.J.
      • Pickett H.A.
      The FANCM-BLM-TOP3A-RMI complex suppresses alternative lengthening of telomeres (ALT).
      ,
      • Sobinoff A.P.
      • Allen J.A.
      • Neumann A.A.
      • Yang S.F.
      • Walsh M.E.
      • Henson J.D.
      • Reddel R.R.
      • Pickett H.A.
      BLM and SLX4 play opposing roles in recombination-dependent replication at human telomeres.
      ,
      • Medves S.
      • Auchter M.
      • Chambeau L.
      • Gazzo S.
      • Poncet D.
      • Grangier B.
      • Verney A.
      • Moussay E.
      • Ammerlaan W.
      • Brisou G.
      • Morjani H.
      • Géli V.
      • Palissot V.
      • Berchem G.
      • Salles G.
      • Wenner T.
      A high rate of telomeric sister chromatid exchange occurs in chronic lymphocytic leukaemia B-cells.
      ,
      • Kim Y.C.
      • Lee M.H.
      • Ryu S.S.
      • Kim J.H.
      • Koo H.S.
      Coaction of DNA topoisomerase IIIα and a RecQ homologue during the germ-line mitosis in Caenorhabditis elegans.
      ,
      • Séguéla-Arnaud M.
      • Crismani W.
      • Larchevêque C.
      • Mazel J.
      • Froger N.
      • Choinard S.
      • Lemhemdi A.
      • Macaisne N.
      • Van Leene J.
      • Gevaert K.
      • De Jaeger G.
      • Chelysheva L.
      • Mercier R.
      Multiple mechanisms limit meiotic crossovers: TOP3α and two BLM homologs antagonize crossovers in parallel to FANCM.
      ,
      • Chaudhury I.
      • Sareen A.
      • Raghunandan M.
      • Sobeck A.
      FANCD2 regulates BLM complex functions independently of FANCI to promote replication fork recovery.
      ).

      Roles of TOP3A at ultrafine anaphase bridges

      Cells expressing defective TOP3A exhibit an increased frequency of ultrafine anaphase bridges (UFBs) (
      • Bizard A.H.
      • Allemand J.F.
      • Hassenkam T.
      • Paramasivam M.
      • Sarlós K.
      • Singh M.I.
      • Hickson I.D.
      PICH and TOP3A cooperate to induce positive DNA supercoiling.
      ,
      • Martin C.A.
      • Sarlós K.
      • Logan C.V.
      • Thakur R.S.
      • Parry D.A.
      • Bizard A.H.
      • Leitch A.
      • Cleal L.
      • Ali N.S.
      • Al-Owain M.A.
      • Allen W.
      • Altmüller J.
      • Aza-Carmona M.
      • Barakat B.A.Y.
      • Barraza-García J.
      • et al.
      Mutations in TOP3A cause a Bloom syndrome-like disorder.
      ). UFBs are thin DNA structures that are stretched between the segregating sister genomes during the anaphase of mitosis (
      • Chan K.L.
      • North P.S.
      • Hickson I.D.
      BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges.
      ,
      • Baumann C.
      • Körner R.
      • Hofmann K.
      • Nigg E.A.
      PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint.
      ,
      • Bizard A.H.
      • Hickson I.D.
      Anaphase: a fortune-teller of genomic instability.
      ). They arise due to the presence of topological entanglements between sister chromatids that fail to be resolved prior to anaphase onset. A variety of topological entanglements give rise to UFBs, such as those arising in replication intermediates, recombination intermediates, and fully catenated DNA (
      • Chan Y.W.
      • Fugger K.
      • West S.C.
      Unresolved recombination intermediates lead to ultra-fine anaphase bridges, chromosome breaks and aberrations.
      ,
      • Chan K.L.
      • Palmai-Pallag T.
      • Ying S.
      • Hickson I.D.
      Replication stress induces sister-chromatid bridging at fragile site loci in mitosis.
      ,
      • Nielsen C.F.
      • Huttner D.
      • Bizard A.H.
      • Hirano S.
      • Li T.N.
      • Palmai-Pallag T.
      • Bjerregaard V.A.
      • Liu Y.
      • Nigg E.A.
      • Wang L.H.C.
      • Hickson I.D.
      PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.
      ,
      • d'Alcontres M.S.
      • Palacios J.A.
      • Mejias D.
      • Blasco M.A.
      TopoIIα prevents telomere fragility and formation of ultra thin DNA bridges during mitosis through TRF1-dependent binding to telomeres.
      ). Hence, the increased frequency of UFBs in TOP3A-defective cells might be at least partly due to the possible roles of this topoisomerase in replication and homologous recombination during interphase. Furthermore, all members of the dissolvasome localize to UFBs, and all of the DNA structures predicted to give rise to UFBs have been shown to be substrates for the dissolvasome complex in vitro, suggesting that the dissolvasome might additionally act in the resolution of these intertwined structures directly during mitosis (Fig. 5) (
      • Sarlós K.
      • Biebricher A.S.
      • Bizard A.H.
      • Bakx J.A.M.
      • Ferreté-Bonastre A.G.
      • Modesti M.
      • Paramasivam M.
      • Yao Q.
      • Peterman E.J.G.
      • Wuite G.J.L.
      • Hickson I.D.
      Reconstitution of anaphase DNA bridge recognition and disjunction.
      ,
      • Chan K.L.
      • North P.S.
      • Hickson I.D.
      BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges.
      ).
      In addition to a possible role in resolving UFBs together with BLM, TOP3A may also act on these structures in conjunction with PICH. Indeed, PICH and TOP3A co-localize on UFBs during the anaphase of mitosis, and depletion of TOP3A or PICH inactivation leads to an increased frequency of the ultrafine anaphase bridges that arise from centromeres (cen-UFBs) (
      • Bizard A.H.
      • Allemand J.F.
      • Hassenkam T.
      • Paramasivam M.
      • Sarlós K.
      • Singh M.I.
      • Hickson I.D.
      PICH and TOP3A cooperate to induce positive DNA supercoiling.
      ,
      • Nielsen C.F.
      • Huttner D.
      • Bizard A.H.
      • Hirano S.
      • Li T.N.
      • Palmai-Pallag T.
      • Bjerregaard V.A.
      • Liu Y.
      • Nigg E.A.
      • Wang L.H.C.
      • Hickson I.D.
      PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.
      ). These are the only UFBs frequently observed during an unperturbed cell cycle and are therefore believed to represent physiological structures (
      • Baumann C.
      • Körner R.
      • Hofmann K.
      • Nigg E.A.
      PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint.
      ). They most likely correspond to fully catenated sister chromatids that are resolved shortly after anaphase onset (
      • Wang L.H.C.
      • Schwarzbraun T.
      • Speicher M.R.
      • Nigg E.A.
      Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation.
      ). Although efficient resolution of cen-UFBs is mediated primarily by TOP2A, it is facilitated by PICH and TOP3A (
      • Bizard A.H.
      • Allemand J.F.
      • Hassenkam T.
      • Paramasivam M.
      • Sarlós K.
      • Singh M.I.
      • Hickson I.D.
      PICH and TOP3A cooperate to induce positive DNA supercoiling.
      ,
      • Nielsen C.F.
      • Huttner D.
      • Bizard A.H.
      • Hirano S.
      • Li T.N.
      • Palmai-Pallag T.
      • Bjerregaard V.A.
      • Liu Y.
      • Nigg E.A.
      • Wang L.H.C.
      • Hickson I.D.
      PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis.
      ). Because the decatenation activity of TOP2 has been proposed to be most efficient on positively supercoiled catenanes, there is the intriguing possibility that PICH and TOP3A catalyze positive supercoiling to facilitate the rapid decatenation of cen-UFBs through stimulation of TOP2A (
      • Marko J.F.
      Coupling of intramolecular and intermolecular linkage complexity of two DNAs.
      ,
      • Baxter J.
      • Sen N.
      • Martínez V.L.
      • De Carandini M.E.
      • Schvartzman J.B.
      • Diffley J.F.X.
      • Aragón L.
      Positive supercoiling of mitotic DNA drives decatenation by topoisomerase II in eukaryotes.
      ,
      • Vologodskii A.
      Unlinking of supercoiled DNA catenanes by type IIA topoisomerases.
      ). In this context and by analogy with the function of prokaryotic reverse gyrases critical to protect DNA against thermal denaturation (
      • Forterre P.
      • Gribaldo S.
      • Gadelle D.
      • Serre M.C.
      Origin and evolution of DNA topoisomerases.
      ,
      • Perugino G.
      • Valenti A.
      • D'amaro A.
      • Rossi M.
      • Ciaramella M.
      Reverse gyrase and genome stability in hyperthermophilic organisms.
      ,
      • Forterre P.
      A hot story from comparative genomics: reverse gyrase is the only hyperthermophile-specific protein.
      ), the positive supercoiling activity of the PICH-TOP3A complex might also contribute to protecting DNA against denaturation when exposed to mitotic spindle forces (
      • Strick T.R.
      • Allemand J.F.
      • Bensimon D.
      • Bensimon A.
      • Croquette V.
      The elasticity of a single supercoiled DNA molecule.
      ). Moreover, although PICH function is most likely restricted to mitosis due to its localization in the cytoplasm during interphase, one cannot exclude the possibility that other topoisomerase-associated complexes catalyze positive supercoiling during interphase. Several scenarios in which a positive supercoiling activity might be necessary can be envisaged. For instance, it might be exploited to down-regulate any process that requires DNA melting for its initiation, such as transcription, replication, and homologous recombination (
      • Baranello L.
      • Wojtowicz D.
      • Cui K.
      • Devaiah B.N.
      • Chung H.J.
      • Chan-Salis K.Y.
      • Guha R.
      • Wilson K.
      • Zhang X.
      • Zhang H.
      • Piotrowski J.
      • Thomas C.J.
      • Singer D.S.
      • Pugh B.F.
      • Pommier Y.
      • Przytycka T.M.
      • Kouzine F.
      • Lewis B.A.
      • Zhao K.
      • Levens D.
      RNA polymerase II regulates topoisomerase 1 activity to favor efficient transcription.
      ,
      • Aze A.
      • Sannino V.
      • Soffientini P.
      • Bachi A.
      • Costanzo V.
      Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.
      ,
      • Fulconis R.
      • Bancaud A.
      • Allemand J.F.
      • Croquette V.
      • Dutreix M.
      • Viovy J.L.
      Twisting and untwisting a single DNA molecule covered by RecA protein.
      ,
      • Cai L.
      • Marquardt U.
      • Zhang Z.
      • Taisey M.J.
      • Chen J.
      Topological testing of the mechanism of homology search promoted by RecA protein.
      ,
      • Wong B.C.
      • Chiu S.K.
      • Chow S.A.
      The role of negative superhelicity and length of homology in the formation of paranemic joints promoted by RecA protein.
      ). Supercoiling could also influence genome architecture at different levels, such as nucleosome dynamics, the formation and dynamics of topologically associated domains, and chromosome condensation (
      • Teves S.S.
      • Henikoff S.
      Transcription-generated torsional stress destabilizes nucleosomes.
      ,
      • Díaz-Ingelmo O.
      • Martínez-García B.
      • Segura J.
      • Valdés A.
      • Roca J.
      DNA topology and global architecture of point centromeres.
      ,
      • Corless S.
      • Gilbert N.
      Effects of DNA supercoiling on chromatin architecture.
      ).

      Possible roles during replication

      Early studies, largely performed in E. coli, suggested that Top3 plays a redundant role in DNA replication with the bacterial Type II topoisomerase, Topo IV (
      • Nurse P.
      • Levine C.
      • Hassing H.
      • Marians K.J.
      Topoisomerase III can serve as the cellular decatenase in Escherichia coli.
      ,
      • DiGate R.J.
      • Marians K.J.
      Identification of a potent decatenating enzyme from Escherichia coli.
      ,
      • Hiasa H.
      • Marians K.J.
      Topoisomerase III, but not topoisomerase I, can support nascent chain elongation during θ-type DNA replication.
      ,
      • Perez-Cheeks B.A.
      • Lee C.
      • Hayama R.
      • Marians K.J.
      A role for topoisomerase III in Escherichia coli chromosome segregation.
      ). It was proposed that TOP3s could participate in decatenation of newly replicated DNA by acting on gapped or nicked precatenanes directly behind the replication fork, a model that was confirmed recently in E. coli (
      • Lee C.M.
      • Wang G.
      • Pertsinidis A.
      • Marians K.J.
      Topoisomerase III acts at the replication fork to remove precatenanes.
      ). However, whether this also occurs in eukaryotic cells is unknown. Besides a role in precatenane removal, the activities of TOP3s could be relevant to other topological aspects associated with DNA replication. For example, in common with any DNA annealing process, the synthesis of a new dsDNA molecule during DNA replication has to be coupled with the de novo formation of topological intertwining to give rise to a double helix. Whereas on the lagging strand this introduction of intertwining can be achieved by free rotation of the Okazaki fragment ends, the synthesis of the leading strand has to be associated with an active introduction of topological linkages. The moderate relaxation activity exhibited by TOP3 may be well-suited to the relaxation of the extreme density of negative supercoils generated on the newly synthesized leading strand, while maintaining a degree of supercoiling compatible with nucleosome assembly. In eukaryotes, replication termination has been proposed to be initiated by convergent fork rotation and stalling resulting from the accumulation of positive supercoils in the short unreplicated segment that can no longer be accessed by TOP1 or TOP2 (
      • Dewar J.M.
      • Budzowska M.
      • Walter J.C.
      The mechanism of DNA replication termination in vertebrates.
      ,
      • Heintzman D.R.
      • Campos L.V.
      • Byl J.A.W.
      • Osheroff N.
      • Dewar J.M.
      Topoisomerase II is crucial for fork convergence during vertebrate replication termination.
      ). The ability of the dissolvasome to resolve late replication intermediates may be ideally suited to processing these structures formed during the latter stages of DNA replication (
      • Suski C.
      • Marians K.J.
      Resolution of converging replication forks by RecQ and topoisomerase III.
      ,
      • Wang J.C.
      Cellular roles of DNA topoisomerases: a molecular perspective.
      ,
      • Fricke W.M.
      • Brill S.J.
      Slx1–Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1-Top3.
      ,
      • Mundbjerg K.
      • Jørgensen S.W.
      • Fredsøe J.
      • Nielsen I.
      • Pedersen J.M.
      • Bentsen I.B.
      • Lisby M.
      • Bjergbaek L.
      • Andersen A.H.
      Top2 and Sgs1-Top3 act redundantly to ensure rDNA replication termination.
      ). Finally, it should be mentioned that the dissolvasome has been previously proposed to play a role in establishment of sister chromatid cohesion via a pathway that depends on the homologous recombination protein Rad51 and independently of other known cohesion pathways (
      • Lai M.S.
      • Seki M.
      • Tada S.
      • Enomoto T.
      Rmi1 functions in S phase-mediated cohesion establishment via a pathway involving the Ctf18-RFC complex and Mrc1.
      ,
      • Lai M.S.
      • Seki M.
      • Ui A.
      • Enomoto T.
      Rmi1, a member of the Sgs1-Top3 complex in budding yeast, contributes to sister chromatid cohesion.
      ). Although highly speculative, it is at least conceivable that the dissolvasome could perform this function by introducing, rather that resolving, entanglements between sister chromatids.

      Roles of mitochondrial TOP3A

      In higher eukaryotes, the TOP3A gene encodes both a nuclear and a mitochondrial isoform, the latter being expressed from an alternative start codon that creates a mitochondrial targeting sequence at the N terminus of the TOP3A polypeptide (
      • Plank J.L.
      • Chu S.H.
      • Pohlhaus J.R.
      • Wilson-Sali T.
      • Hsieh T.S.
      Drosophila melanogaster topoisomerase IIIα preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.
      ,
      • Wang Y.
      • Lyu Y.L.
      • Wang J.C.
      Dual localization of human DNA topoisomerase IIIα to mitochondria and nucleus.
      ) (Fig. 7A). To our knowledge, it is only in Drosophila that the mitochondrial isoform has been genetically ablated. This led to infertility and premature aging, but did not affect organismal viability. These findings indicate that only the nuclear isoform of TOP3A is required during embryonic development, but that mtTOP3 is essential for mtDNA stability (
      • Plank J.L.
      • Chu S.H.
      • Pohlhaus J.R.
      • Wilson-Sali T.
      • Hsieh T.S.
      Drosophila melanogaster topoisomerase IIIα preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.
      ,
      • Tsai H.Z.
      • Lin R.K.
      • Hsieh T.S.
      Drosophila mitochondrial topoisomerase III α affects the aging process via maintenance of mitochondrial function and genome integrity.
      ,
      • Wu J.
      • Feng L.
      • Hsieh T.S.
      Drosophila topo IIIα is required for the maintenance of mitochondrial genome and male germ-line stem cells.
      ).
      Some of the patients described above who harbor TOP3A mutations display severe cardiomyopathy reminiscent of that seen in disorders associated with mitochondrial abnormalities (
      • Martin C.A.
      • Sarlós K.
      • Logan C.V.
      • Thakur R.S.
      • Parry D.A.
      • Bizard A.H.
      • Leitch A.
      • Cleal L.
      • Ali N.S.
      • Al-Owain M.A.
      • Allen W.
      • Altmüller J.
      • Aza-Carmona M.
      • Barakat B.A.Y.
      • Barraza-García J.
      • et al.
      Mutations in TOP3A cause a Bloom syndrome-like disorder.
      ). Furthermore, a compound heterozygous mutation of TOP3A has been identified in an individual with a mitochondrial disorder (
      • Nicholls T.J.
      • Nadalutti C.A.
      • Motori E.
      • Sommerville E.W.
      • Gorman G.S.
      • Basu S.
      • Hoberg E.
      • Turnbull D.M.
      • Chinnery P.F.
      • Larsson N.G.
      • Larsson E.
      • Falkenberg M.
      • Taylor R.W.
      • Griffith J.D.
      • Gustafsson C.M.
      Topoisomerase 3α is required for decatenation and segregation of human mtDNA.
      ). Significantly, the only functional protein expressed in this case carried an amino acid substitution in the TOP3A core catalytic domain (M100V). Cells harboring this defect exhibit perturbed mtDNA segregation and extensive mtDNA rearrangements, which is consistent with a key function of TOP3A in the resolution of hemicatenanes formed specifically during mitochondrial genome replication (
      • Nicholls T.J.
      • Nadalutti C.A.
      • Motori E.
      • Sommerville E.W.
      • Gorman G.S.
      • Basu S.
      • Hoberg E.
      • Turnbull D.M.
      • Chinnery P.F.
      • Larsson N.G.
      • Larsson E.
      • Falkenberg M.
      • Taylor R.W.
      • Griffith J.D.
      • Gustafsson C.M.
      Topoisomerase 3α is required for decatenation and segregation of human mtDNA.
      ). Importantly, none of the core nuclear TOP3A partners (BLM, RMI1, and RMI2; see below for a description of RMI1 and RMI2) has been convincingly shown to reside in mitochondria, suggesting that TOP3A operates independently of the dissolvasome in the mitochondria (
      • Nicholls T.J.
      • Nadalutti C.A.
      • Motori E.
      • Sommerville E.W.
      • Gorman G.S.
      • Basu S.
      • Hoberg E.
      • Turnbull D.M.
      • Chinnery P.F.
      • Larsson N.G.
      • Larsson E.
      • Falkenberg M.
      • Taylor R.W.
      • Griffith J.D.
      • Gustafsson C.M.
      Topoisomerase 3α is required for decatenation and segregation of human mtDNA.
      ,
      • Calvo S.E.
      • Clauser K.R.
      • Mootha V.K.
      MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins.
      ,
      • Rhee H.-W.
      • Zou P.
      • Udeshi N.D.
      • Martell J.D.
      • Mootha V.K.
      • Carr S.A.
      • Ting A.Y.
      Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.
      ).

      Roles of TOP3B in RNA metabolism

      TOP3B is the least studied of the human DNA topoisomerases probably because it is the only eukaryotic nuclear topoisomerase that does not appear to be essential for life (
      • Kwan K.Y.
      • Wang J.C.
      Mice lacking DNA topoisomerase IIIβ develop to maturity but show a reduced mean lifespan.
      ). TOP3B is nonetheless highly relevant for human health, as revealed by studies showing that TOP3B deficiency is linked to the development of neurodevelopmental defects. Indeed, the TOP3B gene resides on chromosome 22q11.2 in the human genome, a region frequently affected by deletions or duplications leading to congenital heart disease, facial malformation, and cognitive dysfunction (
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Kaufman C.S.
      • Genovese A.
      • Butler M.G.
      Deletion of TOP3B is associated with cognitive impairment and facial dysmorphism.
      ,
      • Pires R.
      • Pires L.M.
      • Vaz S.O.
      • Maciel P.
      • Anjos R.
      • Moniz R.
      • Branco C.C.
      • Cabral R.
      • Carreira I.M.
      • Mota-Vieira L.
      Screening of copy number variants in the 22q11.2 region of congenital heart disease patients from the São Miguel Island, Azores, revealed the second patient with a triplication.
      ,
      • Sørensen K.M.
      • Agergaard P.
      • Olesen C.
      • Andersen P.S.
      • Larsen L.A.
      • Østergaard J.R.
      • Schouten J.P.
      • Christiansen M.
      Detecting 22q11.2 deletions by use of multiplex ligation-dependent probe amplification on DNA from neonatal dried blood spot samples.
      ,
      • Wu D.
      • Chen Y.
      • Chen Q.
      • Wang G.
      • Xu X.
      • Peng A.
      • Hao J.
      • He J.
      • Huang L.
      • Dai J.
      Clinical presentation and genetic profiles of Chinese patients with velocardiofacial syndrome in a large referral centre.
      ,
      • Tarsitano M.
      • Ceglia C.
      • Novelli A.
      • Capalbo A.
      • Lombardo B.
      • Pastore L.
      • Fioretti G.
      • Vicari L.
      • Pisanti M.A.
      • Friso P.
      • Cavaliere M.L.
      Microduplications in 22q11.2 and 8q22.1 associated with mild mental retardation and generalized overgrowth.
      ). Consistent with this, copy number variants and de novo mutations in TOP3B have been linked to an increased risk of neurodevelopmental and cognitive disorders, including autism and schizophrenia (
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Ahmad M.
      • Shen W.
      • Li W.
      • Xue Y.
      • Zou S.
      • Xu D.
      • Wang W.
      Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.
      ,
      • Alemany S.
      • Ribasés M.
      • Vilor-Tejedor N.
      • Bustamante M.
      • Sánchez-Mora C.
      • Bosch R.
      • Richarte V.
      • Cormand B.
      • Casas M.
      • Ramos-Quiroga J.A.
      • Sunyer J.
      New suggestive genetic loci and biological pathways for attention function in adult attention-deficit/hyperactivity disorder.
      ,
      • Rosato M.
      • Stringer S.
      • Gebuis T.
      • Paliukhovich I.
      • Li K.W.
      • Posthuma D.
      • Sullivan P.F.
      • Smit A.B.
      • van Kesteren R.E.
      Combined cellomics and proteomics analysis reveals shared neuronal morphology and molecular pathway phenotypes for multiple schizophrenia risk genes.
      ,
      • Daghsni M.
      • Lahbib S.
      • Fradj M.
      • Sayeb M.
      • Kelmemi W.
      • Kraoua L.
      • Kchaou M.
      • Maazoul F.
      • Echebbi S.
      • Ben Ali N.
      • Abdelhak S.
      • M'rad R.
      TOP3B: a novel candidate gene in juvenile myoclonic epilepsy?.
      ). The involvement of TOP3B in cognition is likely conserved across species because TOP3B has been shown to be essential for correct synaptic formation in both mice and flies (
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Ahmad M.
      • Shen W.
      • Li W.
      • Xue Y.
      • Zou S.
      • Xu D.
      • Wang W.
      Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.
      ). These phenotypes, also observed when TOP1 and TOP2B are defective, suggest that TOP3B has a role in releasing topological constraints associated with transcription (
      • McKinnon P.J.
      Topoisomerases and the regulation of neural function.
      ). Such a role is supported by the efficient relaxation activity of TOP3B in vitro, which appears to be specifically targeted to sites of transcription in vivo where TOP3B acts to prevent the accumulation of R-loops (
      • Yang Y.
      • McBride K.M.
      • Hensley S.
      • Lu Y.
      • Chedin F.
      • Bedford M.T.
      Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.
      ,
      • Huang L.
      • Wang Z.
      • Narayanan N.
      • Yang Y.
      Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization.
      ).
      Somewhat surprisingly, given the above discussion, TOP3B has been shown to localize predominantly to the cytosol, where it associates with mRNA within polyribosomes and stress granules (
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Ahmad M.
      • Shen W.
      • Li W.
      • Xue Y.
      • Zou S.
      • Xu D.
      • Wang W.
      Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.
      ). Such a localization suggests a role of TOP3B outside of the nucleus and independent of its ability to interact with DNA and hints at the possible physiological relevance of the RNA topoisomerase activity of TOP3s. Indeed, whereas TOP3A has apparently lost its RNA topoisomerase activity, TOP3B seems to have evolved to specifically process RNA-containing substrates (see below). Although the cellular role of this RNA topoisomerase activity remains to be defined precisely, TOP3B may resolve RNA torsional stress and/or (pseudo-)knots to facilitate the translation of long mRNAs in vivo (
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ).

      Roles of TOP3B in genome stability maintenance

      TOP3Bnull mice have a shorter lifespan, which has been attributed to the development of inflammatory lesions in multiple organs resulting from autoimmunity hyperactivation probably driven by an accumulation of apoptotic cells (
      • Kwan K.Y.
      • Wang J.C.
      Mice lacking DNA topoisomerase IIIβ develop to maturity but show a reduced mean lifespan.
      ,
      • Kwan K.Y.
      • Greenwald R.J.
      • Mohanty S.
      • Sharpe A.H.
      • Shaw A.C.
      • Wang J.C.
      Development of autoimmunity in mice lacking DNA topoisomerase 3β.
      ). TOP3B-defective mice also display a progressive reduction in fecundity over generations due to an increased incidence of aneuploidy in germ cells (
      • Kwan K.Y.
      • Moens P.B.
      • Wang J.C.
      Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase IIIβ.
      ). These observations highlight a role of TOP3B in the long-term maintenance of genomic stability (
      • Kwan K.Y.
      • Greenwald R.J.
      • Mohanty S.
      • Sharpe A.H.
      • Shaw A.C.
      • Wang J.C.
      Development of autoimmunity in mice lacking DNA topoisomerase 3β.
      ). Consistent with this, a recent study identified a homozygous deletion mutation in TOP3B in a patient with a late-onset, bilateral renal cancer (
      • Zhang T.
      • Wallis M.
      • Petrovic V.
      • Challis J.
      • Kalitsis P.
      • Hudson D.F.
      Loss of TOP3B leads to increased R-loop formation and genome instability.
      ). TOP3B-deficient cells exhibit an increased frequency of DNA damage associated with an accumulation of R-loops, but impaired activation of DNA damage response effectors (
      • Yang Y.
      • McBride K.M.
      • Hensley S.
      • Lu Y.
      • Chedin F.
      • Bedford M.T.
      Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.
      ,
      • Zhang T.
      • Wallis M.
      • Petrovic V.
      • Challis J.
      • Kalitsis P.
      • Hudson D.F.
      Loss of TOP3B leads to increased R-loop formation and genome instability.
      ,
      • Mohanty S.
      • Town T.
      • Yagi T.
      • Scheidig C.
      • Kwan K.Y.
      • Allore H.G.
      • Flavell R.A.
      • Shaw A.C.
      Defective p53 engagement after the induction of DNA damage in cells deficient in topoisomerase 3β.
      ). Although one cannot exclude the possibility that TOP3B plays a direct role in DNA replication or repair, this increase in DNA damage may be directly related to the function of TOP3B in preventing R-loop accumulation during transcription. Indeed, R-loops have been shown to represent a threat to genome stability, in particular in proliferating cells, where they can clash with the replication machinery (
      • Yang Y.
      • McBride K.M.
      • Hensley S.
      • Lu Y.
      • Chedin F.
      • Bedford M.T.
      Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.
      ,
      • Huang L.
      • Wang Z.
      • Narayanan N.
      • Yang Y.
      Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization.
      ,
      • Gómez-González B.
      • Aguilera A.
      Transcription-mediated replication hindrance: a major driver of genome instability.
      ).
      All of these findings suggest that TOP3B plays a role during transcription, alongside TOP1 and TOP2. Similar to TOP3A, TOP3B may also be a multifunctional topoisomerase potentially involved in many more cellular processes. For instance, a mitotic/meiotic role of TOP3B has long been speculated, and it was recently reported to intervene in RNAi-mediated heterochromatin formation and transcriptional silencing in Drosophila (
      • Kwan K.Y.
      • Moens P.B.
      • Wang J.C.
      Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase IIIβ.
      ,
      • Lee S.K.
      • Xue Y.
      • Shen W.
      • Zhang Y.
      • Joo Y.
      • Ahmad M.
      • Chinen M.
      • Ding Y.
      • Ku W.L.
      • De S.
      • Lehrmann E.
      • Becker K.G.
      • Lei E.P.
      • Zhao K.
      • Zou S.
      • Sharov A.
      • Wang W.
      Topoisomerase 3β interacts with RNAi machinery to promote heterochromatin formation and transcriptional silencing in Drosophila.
      ,
      • Kobayashi M.
      • Hanai R.
      M phase-specific association of human topoisomerase IIIβ with chromosomes.
      ,
      • Seki T.
      • Seki M.
      • Onodera R.
      • Katada T.
      • Enomoto T.
      Cloning of cDNA encoding a novel mouse DNA topoisomerase III (topo IIIβ) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis.
      ).

      Regulation of TOP3 activity

      The different functions supported by TOP3A and TOPB in vivo suggest that these isoforms serve as nonredundant topoisomerases. Although no systematic, side-by-side comparison of the activities of TOP3A and TOP3B has been performed, they do apparently display marked differences in their substrate preferences. In particular, TOP3B can fully relax a negatively supercoiled plasmid, which strikingly contrasts with the weak relaxation activities of all other TOP3s in general and of TOP3A in particular (
      • Huang L.
      • Wang Z.
      • Narayanan N.
      • Yang Y.
      Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization.
      ,
      • Seki T.
      • Seki M.
      • Onodera R.
      • Katada T.
      • Enomoto T.
      Cloning of cDNA encoding a novel mouse DNA topoisomerase III (topo IIIβ) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis.
      ,
      • Hotoda N.
      • Hanai R.
      Characterization of recombinant human DNA topoisomerase IIIα activity expressed in yeast.
      ,
      • Goulaouic H.
      • Roulon T.
      • Flamand O.
      • Grondard L.
      • Lavelle F.
      • Riou J.F.
      Purification and characterization of human DNA topoisomerase IIIα.
      ). Although these observations are in contradiction with the initial biochemical characterization of Drosophila TOP3B, which seemed to indicate inefficient DNA relaxation, they raise the interesting possibility that this isoform exhibits unique core DNA topoisomerase properties (
      • Wilson T.M.
      • Chen A.D.
      • Hsieh T.
      Cloning and characterization of Drosophila topoisomerase IIIβ: relaxation of hypernegatively supercoiled DNA.
      ). Furthermore, whereas TOP3A has apparently lost the ability to manipulate the topology of RNA substrates, TOP3B seems to have specifically retained the RNA topoisomerase activity conserved within all other TOP3s (
      • Ahmad M.
      • Xu D.
      • Wang W.
      An assay for detecting RNA topoisomerase activity.
      ,
      • Wang H.
      • Di Gate R.J.
      • Seeman N.C.
      An RNA topoisomerase.
      ,
      • DiGate R.J.
      • Marians K.J.
      Escherichia coli topoisomerase III-catalyzed cleavage of RNA.
      ,
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Ahmad M.
      • Xu D.
      • Wang W.
      Type IA topoisomerases can be “magicians” for both DNA and RNA in all domains of life.
      ). It is possible that differences in the biochemical properties of TOP3A and TOP3B are at least partially supported by some subtle divergence within their core topoisomerase domains. However, it is clear that their respective C-terminal domains and obligatory partners play a prominent role in the specialization of activities of these two TOP3 isoforms.

      C-terminal domain

      The activity of the catalytic core of Type IA topoisomerases is often modulated by the presence of a less well-conserved C-terminal domain (Fig. 7A) (
      • Zhang H.L.
      • Malpure S.
      • Li Z.
      • Hiasa H.
      • DiGate R.J.
      The role of the carboxyl-terminal amino acid residues in Escherichia coli DNA topoisomerase III-mediated catalysis.
      ). In yeast TOP3, the C-terminal domain is short, but it has been extended considerably in all TOP3s found in higher eukaryotes. Importantly, TOP3A and TOP3B have distinct C-terminal domains, which are nonetheless relatively well-conserved for each subclass across species, suggesting that their specialization is at least partly determined by this region. In TOP3A, the C-terminal domain is characterized by the presence of at least three zinc-binding motifs. The first, located directly after the core topoisomerase domain, comprises a four-cysteine-zinc motif (Zn-C4) that is involved in ssDNA binding. It is conserved in several Type IA topoisomerases and has been proposed to correspond to a fifth subdomain of the core topoisomerase domain (
      • Viard T.
      • de la Tour C.B.
      Type IA topoisomerases: a simple puzzle?.
      ). Two other zinc-binding regions are defined by tandem glycine-arginine-phenylalanine (GRF) motifs that mediate interaction with nucleic acids and are potentially involved in targeting the different topoisomerases to their favored substrate(s) in vivo and in vitro (
      • Dorn A.
      • Röhrig S.
      • Papp K.
      • Schröpfer S.
      • Hartung F.
      • Knoll A.
      • Puchta H.
      The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates.
      ,
      • Beran-Steed R.K.
      • Tse-Dinh Y.-C.
      The carboxyl terminal domain of Escherichia coli DNA topoisomerase I confers higher affinity to DNA.
      ,
      • Ahumada A.
      • Tse-Dinh Y.C.
      The Zn(II) binding motifs of E. coli DNA topoisomerase I is part of a high-affinity DNA binding domain.
      ). Besides multiple zinc finger motifs, the C-terminal domain of TOP3B harbors arginine-glycine-glycine (RGG) box motifs that are present in certain RNA-binding proteins and are required for TOP3B activity and its function in vivo (Fig. 7A) (
      • Ahmad M.
      • Shen W.
      • Li W.
      • Xue Y.
      • Zou S.
      • Xu D.
      • Wang W.
      Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.
      ). In addition to their nucleic acid–binding properties, these C-terminal domains might also be involved in mediating protein-protein interactions. In particular, the interaction of TOP3B with the Tudor domain–containing 3 protein (TDRD3) is partly mediated via its RGG box motifs. Similarly, the C-terminal domain of Drosophila TOP3A is involved in an interaction with the BLM helicase (
      • Chen S.H.
      • Wu C.H.
      • Plank J.L.
      • Hsieh T.S.
      Essential functions of C terminus of Drosophila topoisomerase IIIα in double Holliday junction dissolution.
      ). Whereas it is clear that the C-terminal domains of TOP3 enzymes are dispensable for their core topoisomerase activity, their ability to mediate interactions with their substrates and protein partners likely plays a key role in specifying cellular functions. Moreover, based on current knowledge, it is not possible to rule out a more direct role for this domain in modulating the biochemical activity of the topoisomerase core domain, such as in the coordination of the successive steps of the catalytic cycle or even in providing directionality to the movement of the T-segment (
      • Ahumada A.
      • Tse-Dinh Y.C.
      The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I.
      ). In the future, it might be particularly important to gain more understanding of the structure of the C-terminal domain of TOP3s, in particular in the context of its association with the core topoisomerase domain.

      The RMI partners of eukaryotic TOP3s

      Eukaryotic TOP3 enzymes form stable heterodimers with conserved RMI (RecQ-mediated genome instability) accessory proteins, with TOP3A and TOP3B each having a specific partner: RMI1 and TDRD3 (Tudor domain–containing 3 protein), respectively (Fig. 7A) (
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ). Both of these RMI proteins comprise a poorly characterized DUF1767 domain and an oligonucleotide-oligosaccharide–binding fold (OB-fold). OB-folds are known to be present in several proteins involved in DNA metabolism, where they mediate interaction with single-stranded nucleic acids and/or with other OB-fold–containing protein domains. These RMI-associated factors are critical for the stability of their cognate topoisomerase in vivo and should, therefore, be considered as obligatory components of the active topoisomerase (
      • Chen C.F.
      • Brill S.J.
      Binding and activation of DNA topoisomerase III by the Rmi1 subunit.
      ,
      • Yang Y.
      • McBride K.M.
      • Hensley S.
      • Lu Y.
      • Chedin F.
      • Bedford M.T.
      Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.
      ,
      • Xu D.
      • Guo R.
      • Sobeck A.
      • Bachrati C.Z.
      • Yang J.
      • Enomoto T.
      • Brown G.W.
      • Hoatlin M.E.
      • Hickson I.D.
      • Wang W.
      RMI, a new OB-fold complex essential for Bloom syndrome protein to maintain genome stability.
      ). This contention is further supported by the structural features of their interaction with the clamp of the core topoisomerase domain of TOP3A or -B, at the hinge located between subdomains II and IV (Fig. 7B) (
      • Goto-Ito S.
      • Yamagata A.
      • Takahashi T.S.
      • Sato Y.
      • Fukai S.
      Structural basis of the interaction between Topoisomerase IIIβ and the TDRD3 auxiliary factor.
      ,
      • Bocquet N.
      • Bizard A.H.
      • Abdulrahman W.
      • Larsen N.B.
      • Faty M.
      • Cavadini S.
      • Bunker R.D.
      • Kowalczykowski S.C.
      • Cejka P.
      • Hickson I.D.
      • Thomä N.H.
      Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1.
      ). RMI1 and TDRD3 are clearly able to modulate the catalytic activity of their cognate topoisomerase, although via different mechanisms. RMI1 promotes the activity of TOP3A on multiple DNA substrates, apparently by stabilizing the cleavage complex in its open state (
      • Bocquet N.
      • Bizard A.H.
      • Abdulrahman W.
      • Larsen N.B.
      • Faty M.
      • Cavadini S.
      • Bunker R.D.
      • Kowalczykowski S.C.
      • Cejka P.
      • Hickson I.D.
      • Thomä N.H.
      Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1.
      ,
      • Cejka P.
      • Plank J.L.
      • Dombrowski C.C.
      • Kowalczykowski S.C.
      Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.
      ,
      • Bizard A.H.
      • Hickson I.D.
      The dissolution of double Holliday junctions.
      ). The crystal structure of truncated versions of RMI1 and TOP3A revealed that RMI1 inserts a loop into the cavity of the topoisomerase domain of TOP3A (
      • Bocquet N.
      • Bizard A.H.
      • Abdulrahman W.
      • Larsen N.B.
      • Faty M.
      • Cavadini S.
      • Bunker R.D.
      • Kowalczykowski S.C.
      • Cejka P.
      • Hickson I.D.
      • Thomä N.H.
      Structural and mechanistic insight into Holliday-junction dissolution by Topoisomerase IIIα and RMI1.
      ,
      • Wang F.
      • Yang Y.
      • Singh T.R.
      • Busygina V.
      • Guo R.
      • Wan K.
      • Wang W.
      • Sung P.
      • Meetei A.R.
      • Lei M.
      Crystal structures of RMI1 and RMI2, two OB-fold regulatory subunits of the BLM complex.
      ). In addition to modulating the dynamic opening and closing of the gate, this association leads to a significant reduction in the size of TOP3A cavity (Fig. 7B). This might enable RMI1 to exert some steric control over the nature of the T-segment, such as to favor single-stranded nucleic acids (
      • Cejka P.
      • Plank J.L.
      • Dombrowski C.C.
      • Kowalczykowski S.C.
      Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.
      ). In contrast, TDRD3 binding to TOP3B does not appear to significantly reduce the size of the topoisomerase cavity (Fig. 7B) (
      • Goto-Ito S.
      • Yamagata A.
      • Takahashi T.S.
      • Sato Y.
      • Fukai S.
      Structural basis of the interaction between Topoisomerase IIIβ and the TDRD3 auxiliary factor.
      ). However, it does exhibit an intrinsic affinity for both ssDNA and ssRNA and has been shown to increase the processivity of the relaxation activity of TOP3B (
      • Siaw G.E.L.
      • Liu I.F.
      • Lin P.Y.
      • Been M.D.
      • Hsieh T.S.
      DNA and RNA topoisomerase activities of Top3β are promoted by mediator protein Tudor domain-containing protein 3.
      ).
      In addition to modulating catalytic activities, the RMI1 and TDRD3 proteins are important for mediating interactions of their type IA topoisomerase partners with additional proteins. For the TOP3A partner RMI1, this involves a second OB-fold that interacts with another OB-fold protein, RMI2. Hence, together, the RMI1-RMI2 heterodimer contains three OB-folds, which mediate the interaction of TOP3A with other DNA-processing enzymes, such as the BLM and FANCM helicases (Fig. 7A) (
      • Meetei A.R.
      • Sechi S.
      • Wallisch M.
      • Yang D.
      • Young M.K.
      • Joenje H.
      • Hoatlin M.E.
      • Wang W.
      A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.
      ,
      • Wang F.
      • Yang Y.
      • Singh T.R.
      • Busygina V.
      • Guo R.
      • Wan K.
      • Wang W.
      • Sung P.
      • Meetei A.R.
      • Lei M.
      Crystal structures of RMI1 and RMI2, two OB-fold regulatory subunits of the BLM complex.
      ,
      • Hoadley K.A.
      • Xue Y.
      • Ling C.
      • Takata M.
      • Wang W.
      • Keck J.L.
      Defining the molecular interface that connects the Fanconi anemia protein FANCM to the Bloom syndrome dissolvasome.
      ,
      • Hoadley K.A.
      • Xu D.
      • Xue Y.
      • Satyshur K.A.
      • Wang W.
      • Keck J.L.
      Structure and cellular roles of the RMI core complex from the bloom syndrome dissolvasome.
      ,
      • Singh T.R.
      • Ali A.M.
      • Busygina V.
      • Raynard S.
      • Fan Q.
      • Du C.H.
      • Andreassen P.R.
      • Sung P.
      • Meetei A.R.
      BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome.
      ). The TOP3B partner, TDRD3, acts as a multiprotein platform characterized by the presence of multiple domains mediating protein-protein interactions with actors in RNA metabolism, such as the fragile X mental retardation protein and the RNA-induced silencing complex (Fig. 7A) (
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ,
      • Lee S.K.
      • Xue Y.
      • Shen W.
      • Zhang Y.
      • Joo Y.
      • Ahmad M.
      • Chinen M.
      • Ding Y.
      • Ku W.L.
      • De S.
      • Lehrmann E.
      • Becker K.G.
      • Lei E.P.
      • Zhao K.
      • Zou S.
      • Sharov A.
      • Wang W.
      Topoisomerase 3β interacts with RNAi machinery to promote heterochromatin formation and transcriptional silencing in Drosophila.
      ). In particular, the Tudor domain of TDRD3 interacts with methylated arginine modifications of histones and RNA polymerase II, which enables the recruitment of TOP3B to transcription start sites and its integration into downstream mRNA metabolism taking place in the cytoplasm (
      • Yang Y.
      • McBride K.M.
      • Hensley S.
      • Lu Y.
      • Chedin F.
      • Bedford M.T.
      Arginine methylation facilitates the recruitment of TOP3B to chromatin to prevent R loop accumulation.
      ,
      • Xu D.
      • Shen W.
      • Guo R.
      • Xue Y.
      • Peng W.
      • Sima J.
      • Yang J.
      • Sharov A.
      • Srikantan S.
      • Yang J.
      • Fox 3rd, D.
      • Qian Y.
      • Martindale J.L.
      • Piao Y.
      • Machamer J.
      • et al.
      Top3β is an RNA topoisomerase that works with fragile X syndrome protein to promote synapse formation.
      ,
      • Stoll G.
      • Pietiläinen O.P.H.
      • Linder B.
      • Suvisaari J.
      • Brosi C.
      • Hennah W.
      • Leppä V.
      • Torniainen M.
      • Ripatti S.
      • Ala-Mello S.
      • Plöttner O.
      • Rehnström K.
      • Tuulio-Henriksson A.
      • Varilo T.
      • Tallila J.
      • et al.
      Deletion of TOP3β, a component of FMRP-containing mRNPs, contributes to neurodevelopmental disorders.
      ). Interestingly, the RGG box motifs of TOP3B are known to be methylated, which promotes an interaction with the Tudor domain of TDRD3 and leads to a stimulation of TOP3B activity (
      • Huang L.
      • Wang Z.
      • Narayanan N.
      • Yang Y.
      Arginine methylation of the C-terminus RGG motif promotes TOP3B topoisomerase activity and stress granule localization.
      ). This indicates that TOP3B and TDRD3 can interact via their respective C-terminal domains in a manner that depends on post-translational modifications, which might have relevance to how DUF1767/OB-fold domain interactions shape the structural organization of TOP3 complexes.

      Conclusion and perspectives

      Anyone who has wrestled with an uncooperative hosepipe while trying to water their beloved rose garden will know how frustrating the topological entanglements of a long polymer can be. If this annoying aspect of home life were as simple to solve as the problems of DNA topology in cells, we would be able simply to cut the hose and rejoin it again just as a topoisomerase does to DNA. Alas, the skill of the topoisomerase far exceeds that of mere humans, and our attempts to emulate the topoisomerase would inevitably end in failure. It is remarkable how few mistakes topoisomerases make and how they have evolved more than one different mechanism to achieve topological transitions, each one being based on a simple concept that nevertheless is extremely challenging to perform rapidly and with high fidelity. Topoisomerases are indeed nature's DNA magicians.
      In humans, supercoiling homeostasis is thought to result from the interplay between supercoiling-inducing processes (primarily transcription) and the supercoiling relaxation activities of DNA topoisomerases. Despite the fact that they are highly conserved enzymes and are often essential for viability, TOP3s are generally not included in this paradigm because they lack the canonical relaxation activity shared by all other DNA topoisomerases. Instead, these unique enzymes are able to efficiently manipulate the topology of a wide range of DNA and RNA substrates and are necessary during most cellular processes associated with maintenance of genomic stability.
      TOP3s have been shown to cooperate with other DNA-processing enzymes to perform complex topological transactions. These illustrate underappreciated aspects of the biochemistry of DNA-processing enzymes. First, some DNA manipulating proteins, such as SNF2 translocases, could exert their physiological role through an active alteration of DNA topology and thereby contribute, alongside DNA topoisomerases, to the regulation of supercoiling homeostasis in vivo. Such activities are de facto ignored when biochemical assays are performed on DNA substrates that are not topologically constrained (such as oligonucleotides). The reintroduction of DNA topology into biochemical assays could reveal that many of the well-known or noncharacterized DNA-processing enzymes directly alter DNA topology through their catalytic activity. Second, the biochemical characterization of TOP3-associated multienzyme complexes has revealed that the canonical activities of a DNA topoisomerase can be modulated in a highly sophisticated fashion by its partners, giving rise to new topological transactions. This opens the interesting possibility that the activities displayed by stand-alone DNA topoisomerases do not reflect the actual diversity of topoisomerase-mediated transactions. We anticipate that future work focusing on the identification and characterization of topoisomerase-associated multienzyme complexes will uncover the many hidden lives that DNA topoisomerases share with their different partners.
      The field of DNA topology was developed nearly 60 years ago, together with the discovery of the double-helical structure of DNA. Nonetheless, our understanding of how DNA supercoiling is regulated and exploited by eukaryotic cells still requires refinement. In particular, the repertoire of topological transactions catalyzed by topoisomerases and non-topoisomerase DNA-processing enzymes is probably more elaborate than is currently envisaged. In this context, it will be essential to develop new methods for monitoring DNA topology in vivo while refining the existing ones, to build up a more detailed and dynamic view of the topological landscape of our genomes. Altogether, these efforts will likely contribute to a renaissance in the field of DNA topology in which DNA topoisomerases are no longer considered as simple swivels that act to release torsional constraints associated with DNA metabolism, but are celebrated as versatile enzymes that promote highly sophisticated cellular functions.

      Acknowledgments

      We thank all members of the Hickson Laboratory for helpful discussions.

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