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Open AccessEditorial

No Time to Relax and Unwind: Exploration of Topoisomerases and a Growing Field of Study

by Joseph E. Deweese and Neil Osheroff

Int. J. Mol. Sci. 2023, 24(17), 13080; https://doi.org/10.3390/ijms241713080 - 23 Aug 2023

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Abstract

With the topoisomerase field in its sixth decade [...] Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

Research

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16 pages, 2665 KiB

Open AccessArticle

Actions of a Novel Bacterial Topoisomerase Inhibitor against Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enhancement of Double-Stranded DNA Breaks

by Soziema E. Dauda, Jessica A. Collins, Jo Ann W. Byl, Yanran Lu, Jack C. Yalowich, Mark J. Mitton-Fry and Neil Osheroff

Int. J. Mol. Sci. 2023, 24(15), 12107; https://doi.org/10.3390/ijms241512107 - 28 Jul 2023

Cited by 1 | Viewed by 1393

Abstract

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported [...] Read more.

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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27 pages, 8829 KiB

Open AccessArticle

Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions

by Martin Bartas, Kristyna Slychko, Jiří Červeň, Petr Pečinka, Donna J. Arndt-Jovin and Thomas M. Jovin

Int. J. Mol. Sci. 2023, 24(13), 10740; https://doi.org/10.3390/ijms241310740 - 27 Jun 2023

Cited by 2 | Viewed by 2338

Abstract

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is [...] Read more.

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the “B-Z-topoII hypothesis” and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments (“Z-flipons”) as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover “conformase” activity on given G(ate) B-DNA segments (“Z-flipins”), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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21 pages, 4658 KiB

Open AccessArticle

StaR Is a Positive Regulator of Topoisomerase I Activity Involved in Supercoiling Maintenance in Streptococcus pneumoniae

by Antonio A. de Vasconcelos Junior, Jose M. Tirado-Vélez, Antonio J. Martín-Galiano, Diego Megias, María-José Ferrándiz, Pablo Hernández, Mónica Amblar and Adela G. de la Campa

Int. J. Mol. Sci. 2023, 24(6), 5973; https://doi.org/10.3390/ijms24065973 - 22 Mar 2023

Cited by 2 | Viewed by 1433

Abstract

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory [...] Read more.

The DNA topoisomerases gyrase and topoisomerase I as well as the nucleoid-associated protein HU maintain supercoiling levels in Streptococcus pneumoniae, a main human pathogen. Here, we characterized, for the first time, a topoisomerase I regulator protein (StaR). In the presence of sub-inhibitory novobiocin concentrations, which inhibit gyrase activity, higher doubling times were observed in a strain lacking staR, and in two strains in which StaR was over-expressed either under the control of the ZnSO₄-inducible P_Zn promoter (strain ΔstaRP_ZnstaR) or of the maltose-inducible P_Mal promoter (strain ΔstaRpLS1ROMstaR). These results suggest that StaR has a direct role in novobiocin susceptibility and that the StaR level needs to be maintained within a narrow range. Treatment of ΔstaRP_ZnstaR with inhibitory novobiocin concentrations resulted in a change of the negative DNA supercoiling density (σ) in vivo, which was higher in the absence of StaR (σ = −0.049) than when StaR was overproduced (σ = −0.045). We have located this protein in the nucleoid by using super-resolution confocal microscopy. Through in vitro activity assays, we demonstrated that StaR stimulates TopoI relaxation activity, while it has no effect on gyrase activity. Interaction between TopoI and StaR was detected both in vitro and in vivo by co-immunoprecipitation. No alteration of the transcriptome was associated with StaR amount variation. The results suggest that StaR is a new streptococcal nucleoid-associated protein that activates topoisomerase I activity by direct protein-protein interaction. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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12 pages, 2886 KiB

Open AccessArticle

Role of the Water–Metal Ion Bridge in Quinolone Interactions with Escherichia coli Gyrase

by Hannah E. Carter, Baylee Wildman, Heidi A. Schwanz, Robert J. Kerns and Katie J. Aldred

Int. J. Mol. Sci. 2023, 24(3), 2879; https://doi.org/10.3390/ijms24032879 - 02 Feb 2023

Cited by 2 | Viewed by 1530

Abstract

Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes—the bacterial type II topoisomerases gyrase and topoisomerase IV—may allow us to rationally design [...] Read more.

Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes—the bacterial type II topoisomerases gyrase and topoisomerase IV—may allow us to rationally design quinolone-based drugs that overcome resistance. As a step toward this goal, we investigated whether the water–metal ion bridge that has been found to mediate the major point of interaction between Escherichia coli topoisomerase IV and Bacillus anthracis topoisomerase IV and gyrase, as well as Mycobacterium tuberculosis gyrase, exists in E. coli gyrase. This is the first investigation of the water–metal ion bridge and its function in a Gram-negative gyrase. Evidence suggests that the water–metal ion bridge does exist in quinolone interactions with this enzyme and, unlike the Gram-positive B. anthracis gyrase, does use both conserved residues (serine and acidic) as bridge anchors. Furthermore, this interaction appears to play a positioning role. These findings raise the possibility that the water–metal ion bridge is a universal point of interaction between quinolones and type II topoisomerases and that it functions primarily as a binding contact in Gram-positive species and primarily as a positioning interaction in Gram-negative species. Future studies will explore this possibility. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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17 pages, 6858 KiB

Open AccessArticle

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase

by Harry Morgan, Magdalena Lipka-Lloyd, Anna J. Warren, Naomi Hughes, John Holmes, Nicolas P. Burton, Eshwar Mahenthiralingam and Ben D. Bax

Int. J. Mol. Sci. 2023, 24(2), 1634; https://doi.org/10.3390/ijms24021634 - 13 Jan 2023

Cited by 8 | Viewed by 2509

Abstract

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA [...] Read more.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water–metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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Review

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15 pages, 2830 KiB

Open AccessReview

To Break or Not to Break: The Role of TOP2B in Transcription

by Ian G. Cowell, John W. Casement and Caroline A. Austin

Int. J. Mol. Sci. 2023, 24(19), 14806; https://doi.org/10.3390/ijms241914806 - 30 Sep 2023

Cited by 2 | Viewed by 1137

Abstract

Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This [...] Read more.

Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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16 pages, 1670 KiB

Open AccessReview

Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes

by Xiaohua Jiang, Lauren A. Fielding, Hunter Davis, William Carroll, Edward C. Lisic and Joseph E. Deweese

Int. J. Mol. Sci. 2023, 24(15), 12010; https://doi.org/10.3390/ijms241512010 - 27 Jul 2023

Cited by 1 | Viewed by 1378

Abstract

Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effects of thiosemicarbazones (TSC) and metal–TSC complexes have been demonstrated to target several biological processes, including DNA metabolism. Human topoisomerases [...] Read more.

Topoisomerases, common targets for anti-cancer therapeutics, are crucial enzymes for DNA replication, transcription, and many other aspects of DNA metabolism. The potential anti-cancer effects of thiosemicarbazones (TSC) and metal–TSC complexes have been demonstrated to target several biological processes, including DNA metabolism. Human topoisomerases were discovered among the molecular targets for TSCs, and metal-chelated TSCs specifically displayed significant inhibition of topoisomerase II. The processes by which metal–TSCs or TSCs inhibit topoisomerases are still being studied. In this brief review, we summarize the TSCs and metal–TSCs that inhibit various types of human topoisomerases, and we note some of the key unanswered questions regarding this interesting class of diverse compounds. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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16 pages, 2857 KiB

Open AccessReview

Telling Your Right Hand from Your Left: The Effects of DNA Supercoil Handedness on the Actions of Type II Topoisomerases

by Jeffrey Y. Jian and Neil Osheroff

Int. J. Mol. Sci. 2023, 24(13), 11199; https://doi.org/10.3390/ijms241311199 - 07 Jul 2023

Cited by 2 | Viewed by 2201

Abstract

Type II topoisomerases are essential enzymes that modulate the topological state of DNA supercoiling in all living organisms. These enzymes alter DNA topology by performing double-stranded passage reactions on over- or underwound DNA substrates. This strand passage reaction generates a transient covalent enzyme–cleaved [...] Read more.

Type II topoisomerases are essential enzymes that modulate the topological state of DNA supercoiling in all living organisms. These enzymes alter DNA topology by performing double-stranded passage reactions on over- or underwound DNA substrates. This strand passage reaction generates a transient covalent enzyme–cleaved DNA structure known as the cleavage complex. Al-though the cleavage complex is a requisite catalytic intermediate, it is also intrinsically dangerous to genomic stability in biological systems. The potential threat of type II topoisomerase function can also vary based on the nature of the supercoiled DNA substrate. During essential processes such as DNA replication and transcription, cleavage complex formation can be inherently more dangerous on overwound versus underwound DNA substrates. As such, it is important to understand the profound effects that DNA topology can have on the cellular functions of type II topoisomerases. This review will provide a broad assessment of how human and bacterial type II topoisomerases recognize and act on their substrates of various topological states. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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18 pages, 2460 KiB

Open AccessReview

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

by Vita Vidmar, Marlène Vayssières and Valérie Lamour

Int. J. Mol. Sci. 2023, 24(4), 3986; https://doi.org/10.3390/ijms24043986 - 16 Feb 2023

Cited by 4 | Viewed by 1857

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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13 pages, 789 KiB

Open AccessReview

A Mini Review of Novel Topoisomerase II Inhibitors as Future Anticancer Agents

by Cosmas O. Okoro and Toluwase Hezekiah Fatoki

Int. J. Mol. Sci. 2023, 24(3), 2532; https://doi.org/10.3390/ijms24032532 - 28 Jan 2023

Cited by 10 | Viewed by 2611

Abstract

Several reviews of inhibitors of topoisomerase II have been published, covering research before 2018. Therefore, this review is focused primarily on more recent publications with relevant points from the earlier literature. Topoisomerase II is an established target for anticancer drugs, which are further [...] Read more.

Several reviews of inhibitors of topoisomerase II have been published, covering research before 2018. Therefore, this review is focused primarily on more recent publications with relevant points from the earlier literature. Topoisomerase II is an established target for anticancer drugs, which are further subdivided into poisons and catalytic inhibitors. While most of the topoisomerase II-based drugs in clinical use are mostly topoisomerase II poisons, their mechanism of action has posed severe concern due to DNA damaging potential, including the development of multi-drug resistance. As a result, we are beginning to see a gradual paradigm shift towards non-DNA damaging agents, such as the lesser studied topoisomerase II catalytic inhibitors. In addition, this review describes some novel selective catalytic topoisomerase II inhibitors. The ultimate goal is to bring researchers up to speed by curating and delineating new scaffolds as the leads for the optimization and development of new potent, safe, and selective agents for the treatment of cancer. Full article

(This article belongs to the Special Issue Advances in Structure, Function and Molecular Targeting of DNA Topoisomerases)

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