Structures and activation mechanism of the Gabija anti-phage system.

Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.

DNA Topoisomerases in Unicellular Pathogens: Structure, Function, and Druggability.

All organisms, including unicellular pathogens, compulsorily possess DNA topoisomerases for successful nucleic acid metabolism. But particular subtypes of topoisomerases exist, in all prokaryotes and in some unicellular eukaryotes, that are absent in higher eukaryotes. Moreover, topoisomerases from pathogenic members of a niche possess some unique molecular architecture and functionalities completely distinct from their nonpathogenic colleagues. This review will highlight the unique attributes associated with the structures and functions of topoisomerases from the unicellular pathogens, with special reference to bacteria and protozoan parasites. It will also summarise the progress made in the domain pertaining to the druggability of these topoisomerases, upon which a future platform for therapeutic development can be successfully constructed.

All tangled up: how cells direct, manage and exploit topoisomerase function.

Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.

Tricking enzymes in living cells: a mechanism-based strategy for design of DNA topoisomerase biosensors.

Most activity-based molecular probes are designed to target enzymes that catalyze the breaking of chemical bonds and the conversion of a unimolecular substrate into bimolecular products. However, DNA topoisomerases are a class of enzymes that alter DNA topology without producing any molecular segments during catalysis, which hinders the development of practical methods for diagnosing these key biomarkers in living cells. Here, we established a new strategy for the effective sensing of the expression levels and catalytic activities of topoisomerases in cell-free systems and human cells. Using our newly designed biosensors, we tricked DNA topoisomerases within their catalytic cycles to switch on fluorescence and resume new rounds of catalysis. Considering that human topoisomerases have been widely recognized as biomarkers for multiple cancers and identified as promising targets for several anticancer drugs, we believe that these DNA-based biosensors and our design strategy would greatly benefit the future development of clinical tools for cancer diagnosis and treatment.

DNA-Topology Simplification by Topoisomerases.

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.

From coins to cancer therapy: Gold, silver and copper complexes targeting human topoisomerases.

Cancer is a complex issue and, even though the prevention basics and therapy have been implemented, it is still the second leading death cause worldwide. With the hope to discover new powerful and safer molecules to fight cancer, many researchers focused their attention on metal-based compounds, starting from the most famous and successfully employed anticancer drug, i.e. cisplatin. The current article aims to report the most recent discoveries about the use of gold, silver and copper complexes as antitumor agents, highlighting their influences on important enzymes, namely human topoisomerases. The latter are fundamental for the cell life and, if overexpressed, strongly implicated in cancer onset and progression. The identification of lead complexes targeting human topoisomerases and gifted with the appropriate chemical and pharmacological properties represents a fecund starting point to obtain new and more effective anticancer molecules.

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.

Transcription-induced supercoiling explains formation of self-interacting chromatin domains in S. pombe.

The question of how self-interacting chromatin domains in interphase chromosomes are structured and generated dominates current discussions on eukaryotic chromosomes. Numerical simulations using standard polymer models have been helpful in testing the validity of various models of chromosome organization. Experimental contact maps can be compared with simulated contact maps and thus verify how good is the model. With increasing resolution of experimental contact maps, it became apparent though that active processes need to be introduced into models to recapitulate the experimental data. Since transcribing RNA polymerases are very strong molecular motors that induce axial rotation of transcribed DNA, we present here models that include such rotational motors. We also include into our models swivels and sites for intersegmental passages that account for action of DNA topoisomerases releasing torsional stress. Using these elements in our models, we show that transcription-induced supercoiling generated in the regions with divergent-transcription and supercoiling relaxation occurring between these regions are sufficient to explain formation of self-interacting chromatin domains in chromosomes of fission yeast (S. pombe).

DNA Supercoiling, Topoisomerases, and Cohesin: Partners in Regulating Chromatin Architecture?

Although our knowledge of chromatin organization has advanced significantly in recent years, much about the relationships between different features of genome architecture is still unknown. Folding of mammalian genomes into spatial domains is thought to depend on architectural proteins, other DNA-binding proteins, and different forms of RNA. In addition, emerging evidence points towards the possibility that the three-dimensional organisation of the genome is controlled by DNA topology. In this scenario, cohesin, CCCTC-binding factor (CTCF), transcription, DNA supercoiling, and topoisomerases are integrated to dictate different layers of genome organization, and the contribution of all four to gene control is an important direction of future studies. In this perspective, we review recent studies that give new insight on how DNA supercoiling shape chromatin structure.

Topoisomerases: Resistance versus Sensitivity, How Far We Can Go?

DNA topoisomerases are ubiquitously present remarkable molecular machines that help in altering topology of DNA in living cells. The crucial role played by these nucleases during DNA replication, transcription, and recombination vis-à-vis less sequence similarity among different species makes topoisomerases unique and attractive targets for different anticancer and antibacterial drugs. However, druggability of topoisomerases by the existing class of molecules is increasingly becoming questationable due to resistance development predominated by mutations in the corresponding genes. The current scenario facing a decline in the development of new molecules further comprises an important factor that may challenge topoisomerase-targeting therapy. Thus, it is imperative to wisely use the existing inhibitors lest with this rapid rate of losing grip over the target we may not go too far. Furthermore, it is important not only to design new molecules but also to develop new approaches that may avoid obstacles in therapies due to multiple resistance mechanisms. This review provides a succinct account of different classes of topoisomerase inhibitors, focuses on resistance acquired by mutations in topoisomerases, and discusses the various approaches to increase the efficacy of topoisomerase inhibitors. In a later section, we also suggest the possibility of using bisbenzimidazoles along with efflux pump inhibitors for synergistic bactericidal effects.

DNA Topoisomerase Inhibitory Activity of Constituents from the Fruits of Illicium verum.

Three new compounds, a sesquilignan (1) and two glucosylated phenylpropanoids (2, 3), and seven known compounds (4-10), were isolated from the fruits of Illicium verum HOOK. FIL. (Illiciaceae). The structures of 1-3 were determined based on one and two dimensional (1D- and 2D-) NMR data and electronic circular dichroism (ECD) spectra analyses. Compounds 3, 5, 6, and 8-10 exhibited potent inhibitory activities against topoisomerase II with IC50 values of 54.6, 25.5, 17.9, 12.1, 0.3 and 1.0 µM, respectively, compared to etoposide, the positive control, with an IC50 of 43.8 µM.

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria.

Type II DNA topoisomerases are divided into two families, IIA and IIB. Types IIA and IIB enzymes share homologous B subunits encompassing the ATP-binding site, but have non-homologous A subunits catalyzing DNA cleavage. Type IIA topoisomerases are ubiquitous in Bacteria and Eukarya, whereas members of the IIB family are mostly present in Archaea and plants. Here, we report the detection of genes encoding type IIB enzymes in which the A and B subunits are fused into a single polypeptide. These proteins are encoded in several bacterial genomes, two bacterial plasmids and one archaeal plasmid. They form a monophyletic group that is very divergent from archaeal and eukaryotic type IIB enzymes (DNA topoisomerase VI). We propose to classify them into a new subfamily, denoted DNA topoisomerase VIII. Bacterial genes encoding a topoisomerase VIII are present within integrated mobile elements, most likely derived from conjugative plasmids. Purified topoisomerase VIII encoded by the plasmid pPPM1a from Paenibacillus polymyxa M1 had ATP-dependent relaxation and decatenation activities. In contrast, the enzyme encoded by mobile elements integrated into the genome of Ammonifex degensii exhibited DNA cleavage activity producing a full-length linear plasmid and that from Microscilla marina exhibited ATP-independent relaxation activity. Topoisomerases VIII, the smallest known type IIB enzymes, could be new promising models for structural and mechanistic studies.

Interfacial inhibitors.

Targeting macromolecular interface is a general mechanism by which natural products inactivate macromolecular complexes by stabilizing normally transient intermediates. Demonstrating interfacial inhibition mechanism ultimately relies on the resolution of drug-macromolecule structures. This review focuses on medicinal drugs that trap protein-DNA complexes by binding at protein-DNA interfaces. It provides proof-of-concept and detailed structural and mechanistic examples for topoisomerase inhibitors and HIV integrase inhibitors. Additional examples of recent interfacial inhibitors for protein-DNA interfaces are provided, as well as prospects for targeting previously 'undruggable' targets including transcription, replication and chromatin remodeling complexes. References and discussion are included for interfacial inhibitors of protein-protein interfaces.

DNA topology: A central dynamic coordinator in chromatin regulation.

Double helical DNA winds around nucleosomes, forming a beads-on-a-string array that further contributes to the formation of high-order chromatin structures. The regulatory components of the chromatin, interacting intricately with DNA, often exploit the topological tension inherent in the DNA molecule. Recent findings shed light on, and simultaneously complicate, the multifaceted roles of DNA topology (also known as DNA supercoiling) in various aspects of chromatin regulation. Different studies may emphasize the dynamics of DNA topological tension across different scales, interacting with diverse chromatin factors such as nucleosomes, nucleic acid motors that propel DNA-tracking processes, and DNA topoisomerases. In this review, we consolidate recent studies and establish connections between distinct scientific discoveries, advancing our current understanding of chromatin regulation mediated by the supercoiling tension of the double helix. Additionally, we explore the implications of DNA topology and DNA topoisomerases in human diseases, along with their potential applications in therapeutic interventions.

Topoisomerases and site-specific recombinases: similarities in structure and mechanism.

The processes of DNA topoisomerization and site-specific recombination are fundamentally similar: DNA cleavage by forming a phospho-protein covalent linkage, DNA topological rearrangement, and DNA ligation coupled with protein regeneration. Type IB DNA topoisomerases are structurally and mechanistically homologous to tyrosine recombinases. Both enzymes nick DNA double helices independent of metal ions, form 3'-phosphotyrosine intermediates, and rearrange the free 5' ends relative to the uncut strands by swiveling. In contrast, serine recombinases generate 5'-phospho-serine intermediates. A 180° relative rotation of the two halves of a 100 kDa terameric serine recombinase and DNA complex has been proposed as the mechanism of strand exchange. Here I propose an alternative mechanism. Interestingly, the catalytic domain of serine recombinases has structural similarity to the TOPRIM domain, conserved among all Type IA and Type II topoisomerases and responsible for metal binding and DNA cleavage. TOPRIM topoisomerases also cleave DNA to generate 5'-phosphate and 3'-OH groups. Based on the existing biochemical data and crystal structures of topoisomerase II and serine recombinases bound to pre- and post-cleavage DNA, I suggest a strand passage mechanism for DNA recombination by serine recombinases. This mechanism is reminiscent of DNA topoisomerization and does not require subunit rotation.

The torsional state of DNA within the chromosome.

Virtually all processes of the genome biology affect or are affected by the torsional state of DNA. Torsional energy associated with an altered twist facilitates or hinders the melting of the double helix, its molecular interactions, and its spatial folding in the form of supercoils. Yet, understanding how the torsional state of DNA is modulated remains a challenging task due to the multiplicity of cellular factors involved in the generation, transmission, and dissipation of DNA twisting forces. Here, an overview of the implication of DNA topoisomerases, DNA revolving motors, and other DNA interactions that determine local levels of torsional stress in bacterial and eukaryotic chromosomes is provided. Particular emphasis is made on the experimental approaches being developed to assess the torsional state of intracellular DNA and its organization into topological domains.

Topological interaction by entangled DNA loops.

We have discovered a new type of interaction between micro- or nanoscale particles that results from the entanglement of strands attached to their surfaces. Self-complementary DNA single strands on a particle can hybridize to form loops. A similar proximal particle can have its loops catenate with those of the first. Unlike conventional thermodynamic interparticle interactions, the catenation interaction is strongly history and protocol dependent, allowing for nonequilibrium particle assembly. The interactions can be controlled by an interesting combination of forces, temperature, light sensitive cross-linking and enzymatic unwinding of the topological links. This novel topological interaction may lead to new materials and phenomena such as particles strung on necklaces, confined motions on designed contours and surfaces, and colloidal Olympic gels.

[DNA-topoisomerases and their functions in cell].

DNA-topoisomerases are sophisticated enzymes controlling DNA topology in cells. A lot of new data concerning the structure and functions of topoisomerases was published recently. In this review authors discuss basic features of the different types of topoisomerases with respect to catalytic mechanism and focus at the involvement of topoisomerases in various DNA-related cellular processes, such as replication, transcription, recombination, chromatin condensation and daughter chromatides partitioning.

Single-molecule measurements of DNA topology and topoisomerases.

Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.

PolY, a transcriptional regulator with ATPase activity, directly activates transcription of polR in polyoxin biosynthesis in Streptomyces cacaoi.

polY, a transcriptional regulatory gene in the polyoxin biosynthetic cluster of Streptomyces cacaoi, was analysed, and its deduced product (PolY) showed amino acid sequence homology to AfsR from Streptomyces coelicolor A3(2). PolY contains an OmpR-like DNA binding domain at its N-terminal and an ATPase domain in the middle of the protein. Disruption of polY abolished polyoxin biosynthesis, which could be restored by the integration of a single copy of polY into the chromosome of the disruption mutant. Transcription of polR, a pathway-specific regulatory gene of polyoxin biosynthesis, was controlled by polY. Electrophoretic mobility shift assay and DNase I protection experiments indicated that PolY bound to the promoter region of polR, and the binding site contained a direct nucleotide repeat typical of Streptomyces antibiotic regulatory protein binding sites. PolY exhibited ATPase activity in vitro. Additionally, binding of ADP/ATPgammaS to ATPase domain triggered the oligomerization of PolY and enhanced its DNA binding activity. Consistently, further experiments in vivo demonstrated that changes of ADP/ATP concentrations significantly affected PolY activity in the cell. These results suggested that the ATPase domain might be a sensor of endogenous pool of ADP/ATP, whose change modulated PolY activity under the physiological conditions.