Alter DNA Topology Research Articles

Alterations in the mechanical properties of single dsDNA molecules, bare or cell-encapsulated, upon exposure to UVA-only radiation and sunlight

Exposure to ultraviolet radiation, which leads to the formation of mutagenic and cytotoxic DNA lesions such as cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4 PPs), can be potentially fatal. The way UVA forms DNA lesions and alters DNA topology and mechanics is still unclear, unlike the cases of UVC and UVB. Herein, Atomic Force Microscopy (AFM) and AFM-based Force Spectroscopy (AFS) have been employed to investigate the topological and mechanical properties of single DNA molecules, bare or E. coli cell-encapsulated, with or without UVA (solar or from UV lamp) treatment. It is observed that both the dsDNA transitions, i.e., ‘B' to stretched ‘S' conformation and melting transition, are lost in UVA dose-dependent manner. Presumably, this is due to formation of the CPDs and 6–4 lesions that form inter-strand cross-links, causing dsDNA strand separation difficult. Gradual reduction in DNA extension length upon prolonged treatment with UVA-only radiation or sunlight (where, 95 % of solar UV is UVA) also indicates formation of the inter-strand cross-links, since such cross-links can reduce DNA flexibility and increase DNA stiffness. Although these observations are common for both bare and cell-encapsulated DNA, the UVA dose at which the distinctive reversible B-S and melting transition faded away varied widely from 240 kJ/m2 (bare DNA) to 900 kJ/m2 (cellular DNA). The UV-induced DNA damage was also evident in observation of increased number of open circular and linearized topologies, as formed due to single-strand and double-strand breaks, respectively, at damage sites, upon combined action of the apurinic/apyrimidinic site-specific endonucleases IV and V. The extent of DNA damage was further quantified by enzyme-linked immunosorbent assay, which is found to be correlated to the single molecule information.

Genome Instability Induced by Topoisomerase Misfunction.

Topoisomerases alter DNA topology by making transient DNA strand breaks (DSBs) in DNA. The DNA cleavage reaction mechanism includes the formation of a reversible protein/DNA complex that allows rapid resealing of the transient break. This mechanism allows changes in DNA topology with minimal risks of persistent DNA damage. Nonetheless, small molecules, alternate DNA structures, or mutations in topoisomerase proteins can impede the resealing of the transient breaks, leading to genome instability and potentially cell death. The consequences of high levels of enzyme/DNA adducts differ for type I and type II topoisomerases. Top1 action on DNA containing ribonucleotides leads to 2-5 nucleotide deletions in repeated sequences, while mutant Top1 enzymes can generate large deletions. By contrast, small molecules that target Top2, or mutant Top2 enzymes with elevated levels of cleavage lead to small de novo duplications. Both Top1 and Top2 have the potential to generate large rearrangements and translocations. Thus, genome instability due to topoisomerase mis-function is a potential pathogenic mechanism especially leading to oncogenic progression. Recent studies support the potential roles of topoisomerases in genetic changes in cancer cells, highlighting the need to understand how cells limit genome instability induced by topoisomerases. This review highlights recent studies that bear on these questions.

Purine-Rich Element Binding Protein Alpha, a Nuclear Matrix Protein, Has a Role in Prostate Cancer Progression.

Solid tumors as well as leukemias and lymphomas show striking changes in nuclear structure including nuclear size and shape, the number and size of nucleoli, and chromatin texture. These alterations have been used in cancer diagnosis and might be related to the altered functional properties of cancer cells. The nuclear matrix (NM) represents the structural composition of the nucleus and consists of nuclear lamins and pore complexes, an internal ribonucleic protein network, and residual nucleoli. In the nuclear microenvironment, the NM is associated with multi-protein complexes, such as basal transcription factors, signaling proteins, histone-modifying factors, and chromatin remodeling machinery directly or indirectly through scaffolding proteins. Therefore, alterations in the composition of NM could result in altered DNA topology and changes in the interaction of various genes, which could then participate in a cascade of the cancer process. Using an androgen-sensitive prostate cancer cell line, LNCaP, and its androgen-independent derivative, LN96, conventional 2D-proteomic analysis of the NM proteins revealed that purine-rich element binding protein alpha (PURα) was detected in the NM proteins and differentially expressed between the cell lines. In this article, we will review the potential role of the molecule in prostate cancer.

Structure-function analysis of the ATPase domain of African swine fever virus topoisomerase.

The ATPase domain of type II topoisomerase provides energy by hydrolyzing ATP and coordinates with the DNA-binding/cleavage domain to drive and control DNA transport. The precise molecular mechanisms of how these domains respond to DNA binding and ATP hydrolysis signals and communicate with each other remain elusive. We determine the first high-resolution crystal structure of the ATPase domain of African swine fever virus (ASFV) topo II in complex with AMPPNP and biochemically investigate its function in ATPase and DNA relaxation activities. Importantly, we find that mutations at three characteristic regions of the ASFV ATPase domain produce parallel effects on the basal/DNA-stimulated ATPase and relaxation activities, implying the tight coupling of the ATP hydrolysis and strand passage process. Therefore, our data provide important implications for understanding the strand passage mechanism of the type II topoisomerase and the structural basis for developing ATPase domain-targeting antivirals against ASFV.

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

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.

Di-indenopyridines as topoisomerase II-selective anticancer agents: Design, synthesis, and structure–activity relationships

Topoisomerases are key molecular enzymes responsible for altering DNA topology, thus they have long been considered as attractive targets for novel chemotherapeutic agents. Topoisomerase type II (Topo II) catalytic inhibitors embrace a fresh perspective meant to get beyond drawbacks caused by topo II poisons, such as cardiotoxicity and secondary malignancies. Based on previously reported 5H-indeno[1,2-b]pyridines, here we presented new twenty-three hybrid di-indenopyridines along with their topo I/IIα inhibitory and antiproliferative activity. Most of the prepared 11-phenyl-diindenopyridines showed negligible topo I inhibitory activity, showing selectivity over topo II. Among the series, we finally selected compound 17, which displayed 100 % topo IIα inhibition at 20 μM concentration and comparable antiproliferative activity against the tested cell lines. Through competitive EtBr displacement assay, cleavable complex assay, and comet assay, compound 17 was finally determined as a non-intercalative catalytic topo IIα inhibitor. The findings in this study highlight the significance of phenolic, halophenyl, thienyl, and furyl groups at the 4-position of the indane ring in the design and synthesis of di-indenopyridines as potent catalytic topo IIα inhibitors with remarkable anticancer effects.

O-GlcNAcylation promotes topoisomerase IIα catalytic activity in breast cancer chemoresistance.

DNA topoisomerase IIα (TOP2A) plays a vital role in replication and cell division by catalytically altering DNA topology. It is a prominent target for anticancer drugs, but clinical efficacy is often compromised due to chemoresistance. In this study, we investigate the role of TOP2A O-GlcNAcylation in breast cancer cells and patient tumor tissues. Our results demonstrate that elevated TOP2A, especially its O-GlcNAcylation, promotes breast cancer malignant progression and resistance to adriamycin (Adm). O-GlcNAcylation at Ser1469 enhances TOP2A chromatin DNA binding and catalytic activity, leading to resistance to Adm in breast cancer cells and xenograft models. Mechanistically, O-GlcNAcylation-modulated interactions between TOP2A and cell cycle regulators influence downstream gene expression and contribute to breast cancer drug resistance. These results reveal a previously unrecognized mechanistic role for TOP2A O-GlcNAcylation in breast cancer chemotherapy resistance and provide support for targeting TOP2A O-GlcNAcylation in cancer therapy.

Dynamic concatenation of ring DNA leads to non-equilibrium viscoelasticity tuned by DNA size and concentration.

Topoisomerases can cleave, twist, untwist and reconnect ring DNA to enable diverse biological processes including DNA replication and repair, as well as the formation of concatenated structures such as kinetoplasts. At the same time, dense solutions of topologically-varying DNA have been shown to exhibit unique viscoelastic properties that can be tuned by DNA concentration, size and topology. Here, we use dynamic light scattering and particle-tracking microrheology to characterize the non-equilibrium viscoelastic properties of dense solutions of ring DNA undergoing dynamic concatenation via the linking and unlinking action of Topoisomerase II. We show that the frequency-dependent viscoelastic properties of these active Olympic ring hydrogels can be precisely tuned by varying the size and concentration of the DNA rings as well as the rate at which Topoisomerase II links and unlinks the DNA. Our work sheds new light on the oft overlooked role of varying viscoelasticity on biological processes that alter DNA topology, including the formation and restructuring of kinetoplasts, and the unwinding of DNA for replication and repair. Future work will build on this bio-inspired platform by incorporating additional enzymes that fragment and ligate DNA for in situ alteration of DNA length.

Small Prokaryotic DNA-Binding Proteins Protect Genome Integrity throughout the Life Cycle.

Genomes of all organisms are persistently threatened by endogenous and exogenous assaults. Bacterial mechanisms of genome maintenance must provide protection throughout the physiologically distinct phases of the life cycle. Spore-forming bacteria must also maintain genome integrity within the dormant endospore. The nucleoid-associated proteins (NAPs) influence nucleoid organization and may alter DNA topology to protect DNA or to alter gene expression patterns. NAPs are characteristically multifunctional; nevertheless, Dps, HU and CbpA are most strongly associated with DNA protection. Archaea display great variety in genome organization and many inhabit extreme environments. As of yet, only MC1, an archaeal NAP, has been shown to protect DNA against thermal denaturation and radiolysis. ssDNA are intermediates in vital cellular processes, such as DNA replication and recombination. Single-stranded binding proteins (SSBs) prevent the formation of secondary structures but also protect the hypersensitive ssDNA against chemical and nuclease degradation. Ionizing radiation upregulates SSBs in the extremophile Deinococcus radiodurans.

Positive supercoiling favors transcription elongation through lac repressor-mediated DNA loops.

Some proteins, like the lac repressor (LacI), mediate long-range loops that alter DNA topology and create torsional barriers. During transcription, RNA polymerase generates supercoiling that may facilitate passage through such barriers. We monitored E. coli RNA polymerase progress along templates in conditions that prevented, or favored, 400 bp LacI-mediated DNA looping. Tethered particle motion measurements revealed that RNA polymerase paused longer at unlooped LacI obstacles or those barring entry to a loop than those barring exit from the loop. Enhanced dissociation of a LacI roadblock by the positive supercoiling generated ahead of a transcribing RNA polymerase within a torsion-constrained DNA loop may be responsible for this reduction in pause time. In support of this idea, RNA polymerase transcribed 6-fold more slowly through looped DNA and paused at LacI obstacles for 66% less time on positively supercoiled compared to relaxed templates, especially under increased tension (torque). Positive supercoiling propagating ahead of polymerase facilitated elongation along topologically complex, protein-coated templates.

Protein with negative surface charge distribution, Bnr1, shows characteristics of a DNA-mimic protein and may be involved in the adaptation of Burkholderia cenocepacia.

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram‐negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5‐fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5‐fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host‐cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three‐dimensional structure revealed BCAS0292 presents a dimeric β‐structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull‐downs with FLAG‐tagged BCAS0292, including the Histone H1‐like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface‐charged regulatory protein 1 (Bnr1), acts as a DNA‐mimic and binds to DNA‐binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

Positive supercoiling facilitates RNAP elongation past protein-mediated DNA loops

Some proteins, such as the lac repressor (LacI), induce the formation of loops that alter DNA topology and generate torsional barriers. The supercoiling generated by elongating RNA polymerase may facilitate passage through such barriers. Tethered particle motion measurements of E. coli RNA polymerase (RNAP) progress along templates in conditions that prevented, or favored, LacI-mediated DNA looping revealed that RNAP paused longer at unlooped LacI obstacles, or those barring entry to a loop, than those barring exit from the loop.

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.Graphical

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.

Topoisomerase II Poisons: Converting Essential Enzymes into Molecular Scissors.

The extensive length, compaction, and interwound nature of DNA, together with its controlled and restricted movement in eukaryotic cells, create a number of topological issues that profoundly affect all of the functions of the genetic material. Topoisomerases are essential enzymes that modulate the topological structure of the double helix, including the regulation of DNA under- and overwinding and the removal of tangles and knots from the genome. Type II topoisomerases alter DNA topology by generating a transient double-stranded break in one DNA segment and allowing another segment to pass through the DNA gate. These enzymes are involved in a number of critical nuclear processes in eukaryotic cells, such as DNA replication, transcription, and recombination, and are required for proper chromosome structure and segregation. However, because type II topoisomerases generate double-stranded breaks in the genetic material, they also are intrinsically dangerous enzymes that have the capacity to fragment the genome. As a result of this dualistic nature, type II topoisomerases are the targets for a number of widely prescribed anticancer drugs. This article will describe the structure and catalytic mechanism of eukaryotic type II topoisomerases and will go on to discuss the actions of topoisomerase II poisons, which are compounds that stabilize DNA breaks generated by the type II enzyme and convert these essential enzymes into "molecular scissors." Topoisomerase II poisons represent a broad range of structural classes and include anticancer drugs, dietary components, and environmental chemicals.

Trapped topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5' end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.

DNA topoisomerases as targets of anticancer drugs

Eukaryotic DNA topoisomerases are ubiquitous nuclear enzymes that alter DNA topology by breaking and rejoining DNA strands. There are two classes of DNA topoisomerases; DNA topoisomerase I introduces a transient single-strand DNA break, while topoisomerase II introduces transient double-strand DNA breaks for each catalytic reaction. Both are important for solving topological problems arising during DNA replication, transcription, recombination and other cellular functions. Recently, scientists have realized the importance of topoisomerases as new therapeutic targets for antibacterial, antifungal, antiparasitic, antiviral and anticancer drugs. The present review focuses on anticancer drugs targeting mammalian DNA topoisomerases, which are named DNA topoisomerase poison. Camptothecin and VM26 (teniposide) are representative DNA topoisomerase poisons that target DNA topoisomerase I and topoisomerase II, respectively. These drugs alter the breakage-reunion reactions of DNA topoisomerases by trapping topoisomerase-DNA cleavable complexes in both the purified system and cultured cells. However, resistance to various DNA topoisomerase poisons has been documented in cancer cells with repect to MDR1 overexpression, reduced topoisomerase levels, drug-resistant mutant topoisomerase, lengthened cell cycle time and altered DNA repair function. A better understanding of the molecular targets for anticancer drugs and the various drug-resistance mechanisms may help us to discover and tailor new drugs for particular drug-resistant tumors.

H-NS Family Proteins Drastically Change Their Targets in Response to the Horizontal Transfer of the Catabolic Plasmid pCAR1.

H-NS family proteins regulate the expression of many genes by preferably binding to AT-rich genomic regions and altering DNA topology. They are found in both bacterial chromosomes and plasmids, and plasmid-encoded H-NS family proteins have sometimes been suggested to act as a molecular backup of the chromosomally encoded ones. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1, which belongs to incompatibility P-7 group. We have investigated the function of Pmr in Pseudomonas putida KT2440, where two H-NS family proteins (TurA and TurB) encoded on the chromosome are expressed predominantly. Previous transcriptome analyses suggested that TurA, TurB, and Pmr cooperatively regulate numerous genes, but the differentially transcribed genes in KT2440ΔturA(pCAR1), KT2440ΔturB(pCAR1), and KT2440(pCAR1Δpmr) compared with those in KT2440(pCAR1) were somewhat different. Here, we performed RNA sequencing analyses to compare the differentially transcribed genes after the deletion of turA or turB in KT2440, and turA, turB or pmr in KT2440(pCAR1). Three pCAR1-free strains (KT2440, KT2440ΔturA, KT2440ΔturB) and four pCAR1-harboring strains [KT2440(pCAR1), KT2440ΔturA(pCAR1), KT2440ΔturB(pCAR1), KT2440(pCAR1Δpmr)], grown until the log and stationary phases, were used. In KT2440, TurA was the major H-NS family protein regulating a large number and wide range of genes, and both TurA and TurB were suggested to functionally compensate each other, particularly during the stationary phase. In KT2440(pCAR1), the numbers of differentially transcribed genes after the deletion of turA or turB drastically increased compared to those in KT2440. Notably, more than half of the differentially transcribed genes in KT2440ΔturA and KT2440ΔturB did not overlap with those in KT2440ΔturA(pCAR1) and KT2440ΔturB(pCAR1). This dynamic change could be explained by the acquisition of pCAR1 itself and the expression of Pmr. After pCAR1 was transferred into the host, TurA and TurB could be detached from the chromosome of KT2440 and they could newly bind to pCAR1. Moreover, Pmr could reconstitute the chromosome-binding heteromeric oligomers which were formed by TurA and TurB. Our study revealed that horizontal transfer of a plasmid changes the transcriptional network of the chromosomally encoded H-NS family proteins.

Increasing the distance between two monomers of topoisomerase II\u03b2 under the action of antitumor agent 4\u03b2-sulfur-(benzimidazole) 4\u2032-demethylepipodophyllotoxin

Topoisomerases II (Top2s) are a group of essential enzymes involved in replication, transcription, chromosome condensation, and segregation via altering DNA topology. The mechanism of the Top2s poisons such as etoposide (VP-16) was reported as stabilizing the Top2-DNA complex and engendering permanent DNA breakage. As the structurally similar compound of VP-16, a novel 4β-sulfur-substituted 4′-demethylepipodophyllotoxin (DMEP) derivative (compound C-Bi) with superior antitumor activity was developed in our previous study. To understand the structural basis of the compound action, the crystal structure (2.54 Å) of human Top2 β-isoform (hTop2β) cleavage complexes stabilized by compound C-Bi was determined. However, compound C-Bi was not visible in the crystal structure. Through the comparison of the structures of hTop2β-DNA-etoposide ternary complex and hTop2β-DNA binary complex, it could be observed that the distance between drug-binding sites Arg503 of the two monomers was 26.62 Å in hTop2β-DNA-etoposide ternary complex and 34.54 Å in hTop2β-DNA binary complex, respectively. Significant twist were observed in the DNA chains of binary complex. It suggested that compound C-Bi played antitumor roles through increasing spacing of hTop2β monomers. The changes in hTop2β structure further caused double changes in the torsional direction and migration distance of the DNA chains, resulting in impeding religation of DNA.

Trapping of the transport-segment DNA by the ATPase domains of a type II topoisomerase

Type II topoisomerases alter DNA topology to control DNA supercoiling and chromosome segregation and are targets of clinically important anti-infective and anticancer therapeutics. They act as ATP-operated clamps to trap a DNA helix and transport it through a transient break in a second DNA. Here, we present the first X-ray crystal structure solved at 2.83 Å of a closed clamp complete with trapped T-segment DNA obtained by co-crystallizing the ATPase domain of S. pneumoniae topoisomerase IV with a nonhydrolyzable ATP analogue and 14-mer duplex DNA. The ATPase dimer forms a 22 Å protein hole occupied by the kinked DNA bound asymmetrically through positively charged residues lining the hole, and whose mutagenesis impacts the DNA decatenation, DNA relaxation and DNA-dependent ATPase activities of topo IV. These results and a side-bound DNA-ParE structure help explain how the T-segment DNA is captured and transported by a type II topoisomerase, and reveal a new enzyme–DNA interface for drug discovery.