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.

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.

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.

A comprehensive overview of ß-carbolines and its derivatives as anticancer agents.

ß-Carboline alkaloids are a family of natural and synthetic products with structural diversity and outstanding antitumor activities. This review summarizes research developments of ß-carboline and its derivatives as anticancer agents, which focused on both natural and synthetic monomers as well as dimers. In addition, the structure-activity relationship (SAR) analysis of ß-carboline monomers and dimers are summarized and mechanism of action of ß-carboline and its derivatives are also presented. A few possible research directions, suggestions and clues for future work on the development of novel ß-carboline-based anticancer agents with improved expected activities and lesser toxicity are also provided.

ß-Carbolines as potential anticancer agents.

ß-Carbolines are indole alkaloids having a tricyclic pyrido[3,4-b]indole ring in their structure. Since the isolation of first ß-carboline from Peganum harmala in 1841, the isolation and synthesis of various ß-carboline derivatives surged in the following centuries. ß-Carboline derivatives due to their widespread availability from natural sources, structural flexibility, quick reactivity and interaction with varied anticancer targets such as DNA (intercalation, groove binding, etc.), enzymes (GPX4, topoisomerases, kinases, etc.) and proteins (tubulin, ABCG2/BRCP1, etc.) have established themselves as promising lead compounds for the synthesis of various anticancer active agents. The current review covers the synthesis and isolation, anticancer activity, mechanism of action and SAR of various ß-carboline containing molecules, its derivatives and congeners.

Debulking of topoisomerase DNA-protein crosslinks (TOP-DPC) by the proteasome, non-proteasomal and non-proteolytic pathways.

Topoisomerases play a pivotal role in ensuring DNA metabolisms during replication, transcription and chromosomal segregation. To manage DNA topology, topoisomerases generate break(s) in the DNA backbone by forming transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and topoisomerase catalytic tyrosyl residues. Topoisomerases have been identified as the cellular targets of a variety of anti-cancer drugs (e.g. topotecan, irinotecan, etoposide and doxorubicin, and antibiotics (e.g. ciprofloxacin and levofloxacin). These drugs, as well as other exogenous and endogenous agents, convert the transient TOPcc into persistent TOPcc, which we refer to as topoisomerase DNA-protein crosslinks (TOP-DPC) that challenge genome integrity and lead to cell death if left unrepaired. Proteolysis of the bulky protein component of TOP-DPC (debulking) is a poorly understood repair process employed across eukaryotes. TOP-DPC proteolysis can be achieved either by the ubiquitin-proteasome pathway (UPP) or by non-proteasomal proteases, which are typified by the metalloprotease SPRTN/WSS1. Debulking of TOP-DPC exposes the phosphotyrosyl bonds, hence enables tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) to access and cleave the bonds. In this review, we focus on current knowledge of the protease pathways for debulking TOP-DPC and highlighting recent advances in understanding the mechanisms regulating the proteolytic repair pathways. We also discuss the avenues that are being exploited to target the proteolytic repair pathways for improving the clinical outcome of topoisomerase inhibitors.

Grid diagrams as tools to investigate knot spaces and topoisomerase-mediated simplification of DNA topology.

Grid diagrams with their relatively simple mathematical formalism provide a convenient way to generate and model projections of various knots. It has been an open question whether these 2D diagrams can be used to model a complex 3D process such as the topoisomerase-mediated preferential unknotting of DNA molecules. We model here topoisomerase-mediated passages of double-stranded DNA segments through each other using the formalism of grid diagrams. We show that this grid diagram-based modeling approach captures the essence of the preferential unknotting mechanism, based on topoisomerase selectivity of hooked DNA juxtapositions as the sites of intersegmental passages. We show that the grid diagram-based approach provides an important, new, and computationally convenient framework for investigating entanglement in biopolymers.

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.

Antileishmanial activity of terpenylquinones on Leishmania infantum and their effects on Leishmania topoisomerase IB.

Leishmania is the aethiological agent responsible for the visceral leishmaniasis, a serious parasite-borne disease widely spread all over the World. The emergence of resistant strains makes classical treatments less effective; therefore, new and better drugs are necessary. Naphthoquinones are interesting compounds for which many pharmacological properties have been described, including leishmanicidal activity. This work shows the antileishmanial effect of two series of terpenyl-1,4-naphthoquinones (NQ) and 1,4-anthraquinones (AQ) obtained from natural terpenoids, such as myrcene and myrceocommunic acid. They were evaluated both in vitro and ex vivo against the transgenic iRFP-Leishmania infantum strain and also tested on liver HepG2 cells to determine their selectivity indexes. The results indicated that NQ derivatives showed better antileishmanial activity than AQ analogues, and among them, compounds with a diacetylated hydroquinone moiety provided better results than their corresponding quinones. Regarding the terpenic precursor, compounds obtained from the monoterpenoid myrcene displayed good antiparasitic efficiency and low cytotoxicity for mammalian cells, whereas those derived from the diterpenoid showed better antileishmanial activity without selectivity. In order to explore their mechanism of action, all the compounds have been tested as potential inhibitors of Leishmania type IB DNA topoisomerases, but only some compounds that displayed the quinone ring were able to inhibit the recombinant enzyme in vitro. This fact together with the docking studies performed on LTopIB suggested the existence of another mechanism of action, alternative or complementary to LTopIB inhibition. In silico druglikeness and ADME evaluation of the best leishmanicidal compounds has shown good predictable druggability.

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.

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.

DNA Related Enzymes as Molecular Targets for Antiviral and Antitumoral Chemotherapy. A Natural Overview of the Current Perspectives.

BACKGROUND: The discovery of new chemotherapeutic agents still remains a continuous goal to achieve. DNA polymerases and topoisomerases act in nucleic acids metabolism modulating different processes like replication, mitosis, damage repair, DNA topology and transcription. It has been widely documented that Polymerases serve as molecular targets for antiviral and antitumoral chemotherapy. Furthermore, telomerase is a ribonucleoprotein with exacerbated activity in most of the tumor cell lines, becoming as an emergent target in Cancer treatment. METHODS: We undertook an exhaustive search of bibliographic databases for peer-reviewed research literature related to the last decade. The characteristics of screened bibliography describe structure activity relationships and show the principal moieties involved. This work tries to summarize the investigation about natural and semi-synthetic products with natural origin with the faculty to inhibit key enzymes that play a crucial role in DNA metabolism. RESULTS: Eighty-five data references were included in this review, showing natural products widely distributed throughout the plant kingdom and their bioactive properties such as tumor growing inhibitory effects, and anti-AIDS activity. CONCLUSION: The findings of this review confirm the importance to find new drugs and biologically active natural products, and their potential medicinally useful benefits.

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.

Discovery of Benzimidazole-Quinolone Hybrids as New Cleaving Agents toward Drug-Resistant Pseudomonas aeruginosa DNA.

A series of benzimidazole-quinolone hybrids as new potential antimicrobial agents were designed and synthesized. Bioactive assays indicated that some of the prepared compounds exhibited potent antibacterial and antifungal activities. Notably, 2-fluorobenzyl derivative 5 b (ethyl 7-chloro-6-fluoro-1-[[1-[(2-fluorophenyl)methyl]benzimidazol-2-yl]methyl]-4-oxo-quinoline-3-carboxylate) showed remarkable antimicrobial activity against resistant Pseudomonas aeruginosa and Candida tropicalis isolated from infected patients. Active molecule 5 b could not only rapidly kill the tested strains, but also exhibit low toxicity toward Hep-2 cells. It was more difficult to trigger the development of bacterial resistance of P. aeruginosa against 5 b than that against norfloxacin. Molecular docking demonstrated that 5 b could effectively bind with topoisomerase IV-DNA complexes, and quantum chemical studies theoretically elucidated the good antimicrobial activity of compound 5 b. Preliminary experimental reaction mechanism exploration suggested that derivative 5 b could not intercalate into DNA isolated from drug-resistant P. aeruginosa, but was able to cleave DNA effectively, which might further block DNA replication to exert powerful bioactivities. In addition, compound 5 b is a promising antibacterial agent with membrane disruption abilities.

Purification of Saccharomyces cerevisiae Homologous Recombination Proteins Dmc1 and Rdh54/Tid1 and a Fluorescent D-Loop Assay.

Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates.

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 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-Damaging Anticancer Drugs - A Perspective for DNA Repair- Oriented Therapy.

DNA-damaging drugs in cancer present two main problems: therapeutic resistance and side effects and both can associate with DNA repair, which can be targeted in cancer therapy. Bleomycin (BLM) induces complex DNA damages, including strand breaks, base loss and 3'-phosphoglycolate (3'PG) residues repaired by several pathways, but 3'PGs must be processed to the 3'-OH ends, usually by tyrosyl-DNA phosphodiesterase 1 (Tdp1). Therefore, targeting Tdp1 can improve anticancer therapy with BLM. Mitomycin C (MMC) produces a variety of adducts with DNA, including inter-strand cross-links (ICLs) and Xeroderma pigmentosum (XP) proteins, including XPG, XPE and XPF can be crucial for the initial stage of ICL repair, so they can be targeted by inhibitors to increase toxicity of MMC in cancer cells. Although these proteins are essential for nucleotide excision repair (NER), their decreased activity may not be fatal in normal cells as almost all NER substrates can be repaired by other pathways. Four-stranded DNA, resulted mainly from guanine quadruplexes (G-4s), are highly overexpressed at the end of telomeres, where they can inhibit telomerase, hence stabilization G-4s at the telomeres ends can hamper proliferation of cancer cells. Quadruplexes are also found in the promoters of genes important for cancer and are resolved by DNA helicases, which can be targeted in cancer along with stabilization of quadruplexes. As cancer cells often have defects in DNA repair pathway(s), they can be subjected by synthetic lethality, with the most promising results with poly(ADP-ribose) polymerase 1 (PARP-1) and DNA-dependent protein kinase, catalytic subunit (DNA-PKCS).

DNA Damage-inducing Compounds: Unraveling their Pleiotropic Effects Using High Throughput Sequencing.

Compounds causing DNA damage have been used widely in molecular biology and some are used as therapeutic agents in cancer therapy. In most cases, their cellular response is pleiotropic, making it challenging to develop these agents efficiently for potential therapeutic use. Furthermore, this means that such compounds can also affect healthy tissues, which is a major drawback for the use in therapy. Thus, dissecting and understanding not only their molecular mode of action, but also identifying all their cellular targets is critical. With the advent of high throughput DNA sequencing technologies our understanding of the genomic targets of such compounds has increased significantly over recent years. This review gives an overview of some well-studied DNA-damaging agents and dissects what is known about their molecular mode of action, their cellular response and use in clinical settings. It then describes how high throughput-sequencing approaches can be used (a) to study DNAdamaging compounds and (b) to gain insight into their biological activity in vivo.