Type IA Topoisomerases Research Articles

Different functional roles of RTR complex factors in DNA repair and meiosis in Arabidopsis and tomato

The RTR (RecQ/Top3/Rmi1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyperrecombination and sensitivity to genotoxins. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: The top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalising knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom.

Type IA Topoisomerases as Targets for Infectious Disease Treatments.

Infectious diseases are one of the main causes of death all over the world, with antimicrobial resistance presenting a great challenge. New antibiotics need to be developed to provide therapeutic treatment options, requiring novel drug targets to be identified and pursued. DNA topoisomerases control the topology of DNA via DNA cleavage–rejoining coupled to DNA strand passage. The change in DNA topological features must be controlled in vital processes including DNA replication, transcription, and DNA repair. Type IIA topoisomerases are well established targets for antibiotics. In this review, type IA topoisomerases in bacteria are discussed as potential targets for new antibiotics. In certain bacterial pathogens, topoisomerase I is the only type IA topoisomerase present, which makes it a valuable antibiotic target. This review will summarize recent attempts that have been made to identify inhibitors of bacterial topoisomerase I as potential leads for antibiotics and use of these inhibitors as molecular probes in cellular studies. Crystal structures of inhibitor–enzyme complexes and more in-depth knowledge of their mechanisms of actions will help to establish the structure–activity relationship of potential drug leads and develop potent and selective therapeutics that can aid in combating the drug resistant bacterial infections that threaten public health.

Mechanism of Type IA Topoisomerases.

Topoisomerases in the type IA subfamily can catalyze change in topology for both DNA and RNA substrates. A type IA topoisomerase may have been present in a last universal common ancestor (LUCA) with an RNA genome. Type IA topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single DNA or RNA strand. Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. Active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a 5′-phosphotyrosine linkage to the cleaved nucleic acid. A divalent ion interaction helps to position the 3′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. In addition to type IA topoisomerase structures observed by X-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type IA topoisomerases to catalyze the change in the topology of the nucleic acid substrates.

Inter‐domain Protein‐Protein Interaction through a Unique α‐Helix in the Hinge Region of E coli Topoisomerase I Leads to the Conformational Change of the Enzyme

E coli topoisomerase I (EcTopI) is a type IA topoisomerase that relaxes negatively supercoiled DNA to prevent the inhibition of vital cellular processes such as transcription. Type IA topoisomerase polypeptide folds into a torus structure to catalyze the breaking and rejoining of a DNA single‐strand coupled with DNA strand passage, thus maintain the DNA topology. Previously elucidated full‐length crystal structure of EcTopI consisting nine domains (D1–D9), bound with an ssDNA to the C‐terminal domain revealed that D6 has an unusual 21‐amino acids insert in the middle of the first β‐strand. A part of the insert (12 amino acids) forms a unique alpha helix (α1), which is present in the vicinity of the N‐terminal domain at the hinge region of D2 and D4. We hypothesized that the C‐terminal element D6 domain interacts with the N‐terminal elements D2 and D4 domains hinge region through this α1‐helix and aids in the separation of D3 from D4 and D1, thus creating an opening to the toroid hole for DNA strand passage in the catalytic cycle. Site‐directed mutagenesis has been employed to create two mutant EcTopI proteins with deletion of D6‐ α1 helix (residues 647–658, Mut‐1) and substitutions of A651G and A655G to perturb α1‐helix structure (Mut‐2). Biochemical analysis of these mutant proteins showed significantly reduced relaxation activity with negatively supercoiled DNA as substrate, and the kinetics of DNA negative supercoil removal by mutants EcTopI showed clear decrease in the rate of relaxation at different time points versus wild‐type EcTopI. To test the effect of these mutations on protein’s thermal stability, thermal shift assay has been carried out and the result suggested that the mutations affected the inter‐domain interactions without significant destabilization of the polypeptide structure as only the first melting peak is shifted to lower temperature but no shift in second peak has been observed compared to wild‐type. Our findings suggest that D6 α1‐helix supports a functional role in the hinge regions between D2 and D4 where its movement (e.g. a push or a pull) as an arm against the hinge will cause the rotation of D2 in respect to D4 leading to the enzyme undergoing a large conformational change required for catalysis. The results of this study have provided a more in‐depth mechanistic insight into the mechanism of type IA topoisomerases that is required for maintaining DNA topology.Support or Funding InformationNIH grant R 01 GM 054226E coli topoI‐Crystal StructureFigure 1Thermal shift assayFigure 2

Mechanistic insights from structure of Mycobacterium smegmatis topoisomerase I with ssDNA bound to both N- and C-terminal domains.

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

New Insights into Structural and Functional Roles of Indole-3-acetic acid (IAA): Changes in DNA Topology and Gene Expression in Bacteria.

Indole-3-acetic acid (IAA) is a major plant hormone that affects many cellular processes in plants, bacteria, yeast, and human cells through still unknown mechanisms. In this study, we demonstrated that the IAA-treatment of two unrelated bacteria, the Ensifer meliloti 1021 and Escherichia coli, harboring two different host range plasmids, influences the supercoiled state of the two plasmid DNAs in vivo. Results obtained from in vitro assays show that IAA interacts with DNA, leading to DNA conformational changes commonly induced by intercalating agents. We provide evidence that IAA inhibits the activity of the type IA topoisomerase, which regulates the DNA topological state in bacteria, through the relaxation of the negative supercoiled DNA. In addition, we demonstrate that the treatment of E. meliloti cells with IAA induces the expression of some genes, including the ones related to nitrogen fixation. In contrast, these genes were significantly repressed by the treatment with novobiocin, which reduces the DNA supercoiling in bacterial cells. Taking into account the overall results reported, we hypothesize that the IAA action and the DNA structure/function might be correlated and involved in the regulation of gene expression. This work points out that checking whether IAA influences the DNA topology under physiological conditions could be a useful strategy to clarify the mechanism of action of this hormone, not only in plants but also in other unrelated organisms.

DNA Topoisomerases of Kinetoplastid Parasites: Brief Overview and Recent Perspectives.

Topoisomerases are a group of enzymes that resolve DNA topological problems and aid in different DNA transaction processes viz. replication, transcription, recombination, etc. inside cells. These proteins accomplish their feats by steps of DNA strand(s) scission, strand passage or rotation and subsequent rejoining activities. Topoisomerases of kinetoplastid parasites have been extensively studied because of their unusual features. The unique presence of heterodimeric Type IB topoisomerase and prokaryotic 'TopA homologue' Type IA topoisomerase in kinetoplastids still generates immense interest among scientists. Moreover, because of their structural dissimilarity with the host enzymes, topoisomerases of kinetoplastid parasites are attractive targets for chemotherapeutic interventions to kill these deadly parasites. In this review, we summarize historical perspectives and recent advances in kinetoplastid topoisomerase research and how these proteins are exploited for drug targeting.

Direct observation of topoisomerase IA gate dynamics.

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site.

We have obtained new crystal structures of Mycobacterium tuberculosis topoisomerase I, including structures with ssDNA substrate bound to the active site, with and without Mg2+ ion present. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition state position for cleavage of a specific phosphodiester linkage. Meanwhile, the enzyme/DNA complex with bound Mg2+ ion may represent the post-transition state for religation in the enzyme's multiple-step DNA relaxation catalytic cycle. The first observation of Mg2+ ion coordinated with the TOPRIM residues and DNA phosphate in a type IA topoisomerase active site allows assignment of likely catalytic role for the metal and draws a comparison to the proposed mechanism for type IIA topoisomerases. The critical function of a strictly conserved glutamic acid in the DNA cleavage step was assessed through site-directed mutagenesis. The functions assigned to the observed Mg2+ ion can account for the metal requirement for DNA rejoining but not DNA cleavage by type IA topoisomerases. This work provides new structural insights into a more stringent requirement for DNA rejoining versus cleavage in the catalytic cycle of this essential enzyme, and further establishes the potential for selective interference of DNA rejoining by this validated TB drug target.

Rv1495 toxin as a model for inhibitors of Mycobacterium tuberculosis DNA topoisomerase I

Topoisomerases overcome topological barriers encountered during DNA replication, transcription, recombination and repair. The genomes of mycobacteria encode only one type IA topoisomerase; Mycobacterium tuberculosis DNA topoisomerase I (Mtb Top I) is thus essential for cellular survival and proliferation, and has been validated as a novel TB drug target. Inhibitors of Mtb Top I may be useful for development of much needed new therapy for drug resistant TB.Rv1495 is a M. tuberculosis toxin that belongs to the MazEF family (MazE is the antitoxin and MazF is the antitoxin), with endoribonuclease activity. Besides being an endoribonuclease, Rv1495 (MazF homolog in M. tuberculosis) toxin has been shown to interact directly with the C‐terminal domain of Mtb Top I for mutual inhibition of catalytic activity. The C‐terminal domains of bacterial topoisomerase I have diverged in evolution with zinc ribbon domains found in most bacterial topoisomerase I as well as human type IA topoisomerases, while mycobacterial topoisomerase I have C‐terminal domains do not bind Zn(II). The mode of Mtb Top I inhibition by Rv1495 could be informative for the design and discovery of small molecule drugs that can target topoisomerase I in M. tuberculosis and pathogenic NTM (non‐tuberculosis mycobacteria).Previous studies have revealed that inhibition of Mtb Top I is due to the direct physical interaction of the N‐terminal domain of Rv1495 toxin with the 33 kDa C‐terminal domain of Mtb Top I. However, no molecular details about this interaction have been reported. We have established an in vivo genetic selection scheme to identify specific amino acid residues in Mtb Top I required for this protein‐protein interaction and inhibition by Rv1495. E. coli AS17 cells have a temperature sensitive mutation in topA coding for topoisomerase I. Recombinant Mtb Top I expressed by plasmid pLICMTOPI can complement the topAts mutation for growth of AS17 at the non‐permissive temperature of 42°C. This complementation is abolished by co‐transformation of AS17 with a second plasmid pBADRv1495 expressing Rv1495. We have constructed a library of pLICMTOPI plasmid with random mutations in Mtb Top I coding sequence. Following transformation of AS17/pBADRv1495, mutated Mtb Top I clones that can complement growth of AS17 at 42°C in the presence of Rv1495 expression have been isolated successfully. These mutants retain the catalytic function of Mtb Top I to support viability at 42°C, but are resistant to inhibition by Rv1495 toxin. The results from analysis of the mutations involved should allow identification of a potential binding site on Mtb Top I for small molecules that can target mycobacteria topoisomerase I specifically.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

An Assay for Detecting RNA Topoisomerase Activity.

RNA topoisomerase activity has recently been detected in multiple Type IA DNA topoisomerases from all three domains of life: bacteria, archaea, and eukarya. Many, but not all, Type IA topoisomerases are found to possess activities for not only DNA, but also RNA, suggesting that they may solve topological problems for both types of nucleic acids. Here we describe a detailed assay used by our group to detect RNA topoisomerase activity for many Type IA topoisomerases. We discuss the strategy, experimental procedures, troubleshooting, and limitations for this assay.

Type IA DNA Topoisomerases: A Universal Core and Multiple Activities.

All the type IA topoisomerases display universal characteristics relying on a core region basically responsible for the transesterification and the strand passage reaction. First limited to the bacterial domain for a long time, these enzymes were further retrieved in Archaea and Eukarya as well. This is representative of an extremely ancient origin, probably due to an inheritance from the RNA world. As remaining evidence, some current topoisomerases IA have retained a RNA topoisomerase activity. Despite the presence of this core region in all of these TopoIAs, some differences exist and are originated from variable regions, located essentially within both extremities, conferring on them their specificities. During the last 2 decades the evidence of multiple activities and dedicated roles highlighted the importance of the topoisomerases IA. It is now obvious that topoisomerases IA are key enzymes involved in the maintenance of the genome stability. The discovery of these new activities was done thanks to the use of more accurate assays, based on new sophisticated DNA substrates.

Synthesis of Hemicatenanes for the Study of Type IA Topoisomerases.

Hemicatenane is a structure that forms when two DNA duplexes are physically linked through a single-stranded crossover. It is proposed to be an intermediate resulting from double Holliday junction (dHJ) dissolution, repair of replication stalled forks and late stage replication. Our previous study has shown that hemicatenane can be synthesized and dissolved in vitro by hyperthermophilic type IA topoisomerases. Here we present the protocol of hemicatenane synthesis and its structure detection by 2D agarose gel electrophoresis. The generated product can be used as a substrate to study the biochemical mechanism of hemicatenane processing reactions.

Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

All cellular organisms encode type IA topoisomerases which catalyze DNA topological changes essential for DNA transactions. However, the kinetics of the reaction catalyzed by these enzymes remains poorly characterized. Here we measured the rapid kinetics of template binding, cleavage and religation by Sso topo III, a type IA topoisomerase from the hyperthermophilic archaeon Sulfolobus solfataricus, by using a novel FRET/PIFE-based method in a stopped-flow spectrometer. We show that Sso topo III bound the template rapidly, and the rate of binding was 2–3 orders of magnitudes higher than that of template cleavage at 25 °C. The rate of template cleavage was favored over that of template religation by the enzyme, and was more so at lower temperatures (25–55 °C). Significant template cleavage [(2.23 ± 0.11) × 10−3 s−1] was observed while little religation was detectable at 25 °C. This is consistent with the presence of a higher activation energy for template religation (41 ± 5 kcal·mol−1) than that for template cleavage (32 ± 1 kcal·mol−1). Our results provide a kinetic interpretation for the ability of Sso topo III to relax negatively supercoiled DNA only at higher temperature and offer clues to the adaptation of the reaction mechanisms of thermophilic enzymes to high temperature.

Type IA topoisomerases can be “magicians” for both DNA and RNA in all domains of life

ABSTRACTTopoisomerases solve critical topological problems in DNA metabolism and have long been regarded as the “magicians” of the DNA world. Here we present views from 2 of our recent studies indicating that Type IA topoisomerases from all domains of life often possess dual topoisomerase activities for both DNA and RNA. In animals, one of the 2 Type IA topoisomerases, Top3β, contains an RNA-binding domain, possesses RNA topoisomerase activity, binds mRNAs, interacts with mRNA-binding proteins, and associates with active mRNA translation machinery. The RNA-binding domain is required for Top3β to bind mRNAs and promote normal neurodevelopment. Top3β forms a highly conserved complex with Tudor-domain-containing 3 (TDRD3), a protein known to interact with translation factors, histones, RNA polymerase II, single stranded DNA and RNA. Top3β requires TDRD3 for its association with the mRNA translation machinery. We suggest that Type IA topoisomerases can be “magicians” for not only DNA, but also RNA; and they may solve topological problems for both nucleic acids in all domains of life. In animals, Top3β-TDRD3 is a dual-activity topoisomerase complex that can act on DNA to stimulate transcription, and on mRNA to promote translation.

An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.

Topoisomerases are enzymes involved in maintaining the topological state of cellular DNA. Despite many structural, biophysical, and biochemical studies, their dynamic characteristics remain poorly understood. Recent single molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events. However, these experiments cannot determine whether the protein remains DNA bound during the pauses or the relationship between domain movements in the protein and topological changes in the DNA. By combining two orthogonal single molecule techniques, we observed that topoisomerase IA is constantly changing conformation and attempting to modify the topology of DNA, but only succeeds in a fraction of the attempts. Thus, its mechanism can be described as a series of DNA strand passage attempts that culminate in a successful relaxation event.

DNA and RNA topoisomerase activities of Top3β are promoted by mediator protein Tudor domain-containing protein 3

Topoisomerase 3β (Top3β) can associate with the mediator protein Tudor domain-containing protein 3 (TDRD3) to participate in two gene expression processes of transcription and translation. Despite the apparent importance of TDRD3 in binding with Top3β and directing it to cellular compartments critical for gene expression, the biochemical mechanism of how TDRD3 can affect the functions of Top3β is not known. We report here sensitive biochemical assays for the activities of Top3β on DNA and RNA substrates in resolving topological entanglements and for the analysis of TDRD3 functions. TDRD3 stimulates the relaxation activity of Top3β on hypernegatively supercoiled DNA and changes the reaction from a distributive to a processive mode. Both supercoil retention assays and binding measurement by fluorescence anisotropy reveal a heretofore unknown preference for binding single-stranded nucleic acids over duplex. Whereas TDRD3 has a structure-specific binding preference, it does not discriminate between DNA and RNA. This unique property for binding with nucleic acids can have an important function in serving as a hub to form nucleoprotein complexes on DNA and RNA. To gain insight into the roles of Top3β on RNA metabolism, we designed an assay by annealing two single-stranded RNA circles with complementary sequences. Top3β is capable of converting two such single-stranded RNA circles into a double-stranded RNA circle, and this strand-annealing activity is enhanced by TDRD3. These results demonstrate that TDRD3 can enhance the biochemical activities of Top3β on both DNA and RNA substrates, in addition to its function of targeting Top3β to critical sites in subcellular compartments.

RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents.

Bacterial topoisomerase functions are required for regulation of DNA supercoiling and overcoming the DNA topological barriers that are encountered during many vital cellular processes. DNA gyrase and topoisomerase IV of the type IIA bacterial topoisomerase family are important clinical targets for antibacterial therapy. Topoisomerase I, belonging to the type IA topoisomerase family, has recently been validated as a potential antitubercular target. The topoisomerase I activity has been shown to be essential for bacterial viability and infection in a murine model of tuberculosis. Mixture-based combinatorial libraries were screened in this study to identify novel bacterial topoisomerase I inhibitors. Using positional-scanning deconvolution, selective small-molecule inhibitors of bacterial topoisomerase I were identified starting from a polyamine scaffold. Antibacterial assays demonstrated that four of these small-molecule inhibitors of bacterial topoisomerase I are bactericidal against Mycobacterium smegmatis and Mycobacterium tuberculosis The MICs for growth inhibition of M. smegmatis increased with overexpression of recombinant M. tuberculosis topoisomerase I, consistent with inhibition of intracellular topoisomerase I activity being involved in the antimycobacterial mode of action.