Type IA Topoisomerases Research Articles

Structural insights into human topoisomerase 3β DNA and RNA catalysis and nucleic acid gate dynamics

Type IA topoisomerases (TopoIAs) are present in all living organisms. They resolve DNA/RNA catenanes, knots and supercoils by breaking and rejoining single-stranded DNA/RNA segments and allowing the passage of another nucleic acid segment through the break. Topoisomerase III-β (TOP3B), the only RNA topoisomerase in metazoans, promotes R-loop disassembly and translation of mRNAs. Defects in TOP3B lead to severe neurological diseases. We present a series of cryo-EM structures of human TOP3B with its cofactor TDRD3 during cleavage and rejoining of DNA or RNA, thus elucidating the roles of divalent metal ions and key enzyme residues in each step of the catalytic cycle. We also obtained the structure of an open-gate configuration that addresses the long-standing question of the strand-passage mechanism. Our studies reveal how TOP3B catalyzes both DNA and RNA relaxation, while TOP3A acts only on DNA.

Insights into the DNA and RNA Interactions of Human Topoisomerase III Beta Using Molecular Dynamics Simulations.

Human topoisomerase III beta (hTOP3B) is the only topoisomerase in the human cell that can act on both DNA and RNA substrates. Recent findings have emphasized the physiological importance of hTOP3B and consolidated it as a valuable drug target for antiviral and anticancer therapeutics. Although type IA topoisomerases of different organisms have been studied over the years, the step-by-step interaction of hTOP3B and nucleic acid substrates is still not well understood. Due to the lack of hTOP3B-RNA structures as well as DNA/RNA covalent complexes, computational investigations have been limited. In our study, we utilized molecular dynamics (MD) simulations to study the interactions between hTOP3B and nucleic acids to get a closer look into the residues that play a role in binding DNA or RNA and facilitate catalysis, along with the differences and similarities when hTOP3B interacts with DNA compared to RNA. For this, we generated multiple models of hTOP3B complexed with DNA and RNA sequences using the hTOP3B crystal structure and 8-mer single-stranded DNA and RNA sequences. These models include both covalent and noncovalent complexes, which are then subjected to MD simulations and analyzed. Our findings highlight the complexes' stability, sequence preference, and interactions of the binding pocket residues with different nucleotides. Our work demonstrates that hTOP3B forms stable complexes with both DNA and RNA and provides a better understanding of the enzyme's interaction with different nucleic acid substrate sequences.

Purification of Recombinant E. Coli Topoisomerase III for Structure-Based Drug Design Using Protein Crystallization

Type IA Topoisomerases are ubiquitous enzymes found throughout all life forms and species. These topoisomerases relieve the topographical constrains formed by DNA during processes like replication and transcription via a cleavage-religation mechanism performed through a catalytically active tyrosine residue in the primary structure of the enzyme. E. coli Topoisomerase III (EtopIII) is a type of Type IA topoisomerase, and its main function in the cell is as a decatenase, which means that it unlinks circular or intertwined pieces of genetic material and creates two unlinked segments of DNA from a singular linked chain. Structure-based determination of the enzyme’s three-dimensional structure via crystallization could be beneficial in finding a novel antibiotic drug that is able to inhibit this enzyme in vivo, a solution that is sorely needed in a world where antibiotic-resistant bacteria are an ever-growing problem. The crystallization of the enzyme can be undertaken by truncating a part of the C-terminal of the enzyme that makes it harder for it to crystallize and introduce a DNA/RNA ligand to observe its binding interactions with said substrate. For this to happen, the truncated recombinant mutant must be purified and concentrated to desirable levels for crystallization. The results of the experiment show that out of 4L of bacterial starting culture, a total of 34.57 mg of protein was able to be purified. This protein was then sent for crystallization experiments, of which results are still pending, but preliminary results indicate that the protein quickly aggregates in one of the RNA ligand conditions, which elicits a change in protocol for future purifications. Furthermore, results from a relaxation assay indicate that the protein exhibits some relaxation activity even after the truncation of the C-terminal chain. These results could expand the knowledge of this enzyme for future structure-based drug development.

Variation of Structure and Cellular Functions of Type IA Topoisomerases across the Tree of Life.

Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.

Abstract 1656 Identification of a Drug Binding Pocket in Type IA Topoisomerases

Abstract 1656 Identification of a Drug Binding Pocket in Type IA Topoisomerases

Mycobacterial type IA topoisomerase suffer energetically after C-terminal deletion

Mycobacterial type IA topoisomerase suffer energetically after C-terminal deletion

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a β-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length andis dispensable for DNA supercoiling, the β-bulge loop is required for supercoiling activity. It has previously been shown that the β-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β-bulge loop as a minimal latch is reported. It is shown that the β-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli.

The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.

Unraveling topoisomerase IA gate dynamics in presence of PPEF and its preclinical evaluation against multidrug-resistant pathogens

Type IA topoisomerases maintain DNA topology by cleaving ssDNA and relaxing negative supercoils. The inhibition of its activity in bacteria prevents the relaxation of negative supercoils, which in turn impedes DNA metabolic processes leading to cell death. Using this hypothesis, two bisbenzimidazoles, PPEF and BPVF are synthesized, selectively inhibiting bacterial TopoIA and TopoIII. PPEF stabilizes the topoisomerase and topoisomerase-ssDNA complex, acts as an interfacial inhibitor. PPEF display high efficacy against ~455 multi-drug resistant gram positive and negative bacteria. To understand molecular mechanism of inhibition of TopoIA and PPEF, accelerated MD simulation is carried out, and results suggested that PPEF binds, stabilizes the closed conformation of TopoIA with –6Kcal/mol binding energy and destabilizes the binding of ssDNA. The TopoIA gate dynamics model can be used as a tool to screen TopoIA inhibitors as therapeutic candidates. PPEF and BPVF cause cellular filamentation and DNA fragmentation leading to bacterial cell death. PPEF and BPVF show potent efficacy against systemic and neutropenic mouse models harboring E. coli, VRSA, and MRSA infection without cellular toxicity.

What's on the Other Side of the Gate: A Structural Perspective on DNA Gate Opening of Type IA and IIA DNA Topoisomerases.

DNA topoisomerases have an essential role in resolving topological problems that arise due to the double-helical structure of DNA. They can recognise DNA topology and catalyse diverse topological reactions by cutting and re-joining DNA ends. Type IA and IIA topoisomerases, which work by strand passage mechanisms, share catalytic domains for DNA binding and cleavage. Structural information has accumulated over the past decades, shedding light on the mechanisms of DNA cleavage and re-ligation. However, the structural rearrangements required for DNA-gate opening and strand transfer remain elusive, in particular for the type IA topoisomerases. In this review, we compare the structural similarities between the type IIA and type IA topoisomerases. The conformational changes that lead to the opening of the DNA-gate and strand passage, as well as allosteric regulation, are discussed, with a focus on the remaining questions about the mechanism of type IA topoisomerases.

Measuring the effects of C-terminal deletion in mycobacterial type IA topoisomerases using magnetic tweezers.

In the cell, protein replication and transcription are processes that introduce supercoils to DNA. Regulation of this DNA topology is handled by enzymes called DNA topoisomerases. Type IA topoisomerases resolve supercoils in discrete steps by cleaving a single strand of duplex DNA, creating a protein mediated DNA gate, and passing the opposing strand through the opening. The mycobacteria genus of topoisomerases has been less well-studied than the prototypical type IA topoisomerases of E. coli. Two enzymes of interest are Mycobacterium tuberculosis topoisomerase IA (mtbTopIA) and Mycobacterium smegmatis topoisomerase IA (msmTopIA). Mycobacteria type IA topoisomerases have DNA-binding C-terminal domains that are involved in passing the second DNA strand of a melted duplex through the DNA gate. Because these C-terminal domains are unique, they are potential targets for highly specific antibiotics. Using single-molecule magnetic tweezers (MT), we have characterized the relaxation behavior of wild type msmTopIA as well as variants that have had domains from their C-termini removed by mutagenesis. We report clear differences in behavior between the enzymes studied, as well as provide further exploration regarding the purpose and mechanism of the C-termini of these enzymes. In addition, we report that our results agree with those done previously in ensemble assays.

R-loops, type I topoisomerases and cancer.

R-loops are abundant and dynamic structures ubiquitously present in human cells both in the nuclear and mitochondrial genomes. They form in cis in the wake of transcription complexes and in trans apart from transcription complexes. In this review, we focus on the relationship between R-loops and topoisomerases, and cancer genomics and therapies. We summarize the topological parameters associated with the formation and resolution of R-loops, which absorb and release high levels of genomic negative supercoiling (Sc-). We review the deleterious consequences of excessive R-loops and rationalize how human type IA (TOP3B) and type IB (TOP1) topoisomerases regulate and resolve R-loops in coordination with helicase and RNase H enzymes. We also review the drugs (topoisomerase inhibitors, splicing inhibitors, G4 stabilizing ligands) and cancer predisposing genes (BRCA1/2, transcription, and splicing genes) known to induce R-loops, and whether stabilizing R-loops and thereby inducing genomic damage can be viewed as a strategy for cancer treatment.

The interaction between transport-segment DNA and topoisomerase IA-crystal structure of MtbTOP1 in complex with both G- and T-segments.

Each catalytic cycle of type IA topoisomerases has been proposed to comprise multistep reactions. The capture of the transport-segment DNA (T-segment) into the central cavity of the N-terminal toroidal structure is an important action, which is preceded by transient gate-segment (G-segment) cleavage and succeeded by G-segment religation for the relaxation of negatively supercoiled DNA and decatenation of DNA. The T-segment passage in and out of the central cavity requires significant domain-domain rearrangements, including the movement of D3 relative to D1 and D4 for the opening and closing of the gate towards the central cavity. Here we report a direct observation of the interaction of a duplex DNA in the central cavity of a type IA topoisomerase and its associated domain-domain conformational changes in a crystal structure of a Mycobacterium tuberculosis topoisomerase I complex that also has a bound G-segment. The duplex DNA within the central cavity illustrates the non-sequence-specific interplay between the T-segment DNA and the enzyme. The rich structural information revealed from the novel topoisomerase-DNA complex, in combination with targeted mutagenesis studies, provides new insights into the mechanism of the topoisomerase IA catalytic cycle.

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4.

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb's toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM).

Structural and biochemical basis for DNA and RNA catalysis by human Topoisomerase 3β

In metazoans, topoisomerase 3β (TOP3B) regulates R-loop dynamics and mRNA translation, which are critical for genome stability, neurodevelopment and normal aging. As a Type IA topoisomerase, TOP3B acts by general acid-base catalysis to break and rejoin single-stranded DNA. Passage of a second DNA strand through the transient break permits dissipation of hypernegative DNA supercoiling and catenation/knotting. Additionally, hsTOP3B was recently demonstrated as the human RNA topoisomerase, required for normal neurodevelopment and proposed to be a potential anti-viral target upon RNA virus infection. Here we elucidate the biochemical mechanisms of human TOP3B. We delineate the roles of divalent metal ions, and of a conserved Lysine residue (K10) in the differential catalysis of DNA and RNA. We also demonstrate that three regulatory factors fine-tune the catalytic performance of TOP3B: the TOP3B C-terminal tail, its protein partner TDRD3, and the sequence of its DNA/RNA substrates.

Microbial Type IA Topoisomerase C-Terminal Domain Sequence Motifs, Distribution and Combination.

Type IA topoisomerases have highly conserved catalytic N-terminal domains for the cleaving and rejoining of a single DNA/RNA strand that have been extensively characterized. In contrast, the C-terminal region has been less covered. Two major types of small tandem C-terminal domains, Topo_C_ZnRpt (containing C4 zinc finger) and Topo_C_Rpt (without cysteines) were initially identified in Escherichia coli and Mycobacterium tuberculosis topoisomerase I, respectively. Their structures and interaction with DNA oligonucleotides have been revealed in structural studies. Here, we first present the diverse distribution and combinations of these two structural elements in various bacterial topoisomerase I (TopA). Previously, zinc fingers have not been seen in type IA topoisomerases from well-studied fungal species within the phylum Ascomycota. In our extended studies of C-terminal DNA-binding domains, the presence of zf-GRF and zf-CCHC types of zinc fingers in topoisomerase III (Top3) from fungi species in many phyla other than Ascomycota has drawn our attention. We secondly analyze the distribution and combination of these fungal zf-GRF- and zf-CCHC-containing domains. Their potential structures and DNA-binding mechanism are evaluated. The highly diverse arrangements and combinations of these DNA/RNA-binding domains in microbial type IA topoisomerase C-terminal regions have important implications for their interactions with nucleic acids and protein partners as part of their physiological functions.

Human topoisomerases and their roles in genome stability and organization.

Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA–protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer.

Exploration of topoisomerase IA gate dynamics reveals an additional widened state

E. coli topoisomerase I, a type IA topoisomerase, is believed to be the main enzyme responsible forrelaxing negative supercoils in-vivo via a strand passage mechanism. Additionally, in-vitro studies haveestablished that the enzyme in also able to (de)catenate two double stranded DNA molecules via thesame mechanism, provided that one of them contains a nick. Despite the fact that all evidence pointedtowards the existence of a protein-mediated gate as a transition state, the actual gate opening eventwas only recently directly observed, allowing access to the study of the gate dynamics.

Single-molecule characterization of topoisomerase I and mutations using magnetic tweezers

Processes such as protein transcription and replication produce torsional strain on DNA, resulting in supercoiling. A group of enzymes called topoisomerases (topo) are responsible for resolving these supercoils and maintaining the correct DNA topology in the cell. The type IA class of topoisomerases, cleave a strand of duplex DNA and pass a second strand through the break to relieve the torsional strain on DNA. The prototypical type IA topoisomerase, topo I of E. coli have been well characterized.

Controlling the topology of mammalian mitochondrial DNA.

The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.