DNA Catenanes Research Articles

The SUMO-NIP45 pathway processes toxic DNA catenanes to prevent mitotic failure.

SUMOylation regulates numerous cellular processes, but what represents the essential functions of this protein modification remains unclear. To address this, we performed genome-scale CRISPR-Cas9-based screens, revealing that the BLM-TOP3A-RMI1-RMI2 (BTRR)-PICH pathway, which resolves ultrafine anaphase DNA bridges (UFBs) arising from catenated DNA structures, and the poorly characterized protein NIP45/NFATC2IP become indispensable for cell proliferation when SUMOylation is inhibited. We demonstrate that NIP45 and SUMOylation orchestrate an interphase pathway for converting DNA catenanes into double-strand breaks (DSBs) that activate the G2 DNA-damage checkpoint, thereby preventing cytokinesis failure and binucleation when BTRR-PICH-dependent UFB resolution is defective. NIP45 mediates this new TOP2-independent DNA catenane resolution process via its SUMO-like domains, promoting SUMOylation of specific factors including the SLX4 multi-nuclease complex, which contributes to catenane conversion into DSBs. Our findings establish that SUMOylation exerts its essential role in cell proliferation by enabling resolution of toxic DNA catenanes via nonepistatic NIP45- and BTRR-PICH-dependent pathways to prevent mitotic failure.

DNA-Stimulated Liquid-Liquid phase separation by eukaryotic topoisomerase ii modulates catalytic function.

Type II topoisomerases modulate chromosome supercoiling, condensation, and catenation by moving one double-stranded DNA segment through a transient break in a second duplex. How DNA strands are chosen and selectively passed to yield appropriate topological outcomes - for example, decatenation vs. catenation - is poorly understood. Here, we show that at physiological enzyme concentrations, eukaryotic type IIA topoisomerases (topo IIs) readily coalesce into condensed bodies. DNA stimulates condensation and fluidizes these assemblies to impart liquid-like behavior. Condensation induces both budding yeast and human topo IIs to switch from DNA unlinking to active DNA catenation, and depends on an unstructured C-terminal region, the loss of which leads to high levels of knotting and reduced catenation. Our findings establish that local protein concentration and phase separation can regulate how topo II creates or dissolves DNA links, behaviors that can account for the varied roles of the enzyme in supporting transcription, replication, and chromosome compaction.

Recognition of DNA Supercoil Handedness during Catenation Catalyzed by Type II Topoisomerases.

Although the presence of catenanes (i.e., intermolecular tangles) in chromosomal DNA stabilizes interactions between daughter chromosomes, a lack of resolution can have serious consequences for genomic stability. In all species, from bacteria to humans, type II topoisomerases are the enzymes primarily responsible for catenating/decatenating DNA. DNA topology has a profound influence on the rate at which these enzymes alter the superhelical state of the double helix. Therefore, the effect of supercoil handedness on the ability of human topoisomerase IIα and topoisomerase IIβ and bacterial topoisomerase IV to catenate DNA was examined. Topoisomerase IIα preferentially catenated negatively supercoiled over positively supercoiled substrates. This is opposite to its preference for relaxing (i.e., removing supercoils from) DNA and may prevent the enzyme from tangling the double helix ahead of replication forks and transcription complexes. The ability of topoisomerase IIα to recognize DNA supercoil handedness during catenation resides in its C-terminal domain. In contrast to topoisomerase IIα, topoisomerase IIβ displayed little ability to distinguish DNA geometry during catenation. Topoisomerase IV from three bacterial species preferentially catenated positively supercoiled substrates. This may not be an issue, as these enzymes work primarily behind replication forks. Finally, topoisomerase IIα and topoisomerase IV maintain lower levels of covalent enzyme-cleaved DNA intermediates with catenated over monomeric DNA. This allows these enzymes to perform their cellular functions in a safer manner, as catenated daughter chromosomes may be subject to stress generated by the mitotic spindle that could lead to irreversible DNA cleavage.

Processing DNA lesions during mitosis to prevent genomic instability.

Failure of cells to process toxic double-strand breaks (DSBs) constitutes a major intrinsic source of genome instability, a hallmark of cancer. In contrast with interphase of the cell cycle, canonical repair pathways in response to DSBs are inactivated in mitosis. Although cell cycle checkpoints prevent transmission of DNA lesions into mitosis under physiological condition, cancer cells frequently display mitotic DNA lesions. In this review, we aim to provide an overview of how mitotic cells process lesions that escape checkpoint surveillance. We outline mechanisms that regulate the mitotic DNA damage response and the different types of lesions that are carried over to mitosis, with a focus on joint DNA molecules arising from under-replication and persistent recombination intermediates, as well as DNA catenanes. Additionally, we discuss the processing pathways that resolve each of these lesions in mitosis. Finally, we address the acute and long-term consequences of unresolved mitotic lesions on cellular fate and genome stability.

Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions.

Invited for the cover of this issue are Prof. Takashi Morii and co-workers at Kyoto University and Ewha Womans University. The cover image depicts the graphical design and atomic force microscopic (AFM) images of the synthesized topologically-interlocked DNA catenane and rotaxanes inside a frame-shaped DNA origami. Read the full text of the article at 10.1002/chem.202200108.

Kinetics of topological transitions in DNA mediated by topoisomerases and recombinases

Topological properties of DNA (loop formation, supercoiling, knotting, and catenation) play critical roles in biological regulation and genome organization. The influence of DNA topology spans an enormous range of length scales: from the base-pair level to large chromosomal domains. Although there is abundant evidence that DNA topology and DNA supercoiling, in particular, constitutes an important mechanism for exerting “action at a distance” in genomes, we lack effective and generalizable polymer-based models for investigating the absolute and relative rates of DNA topology-dependent processes.

Functional Nucleic Acids for Pathogenic Bacteria Detection.

Pathogens have long presented a significant threat to human lives, and hence the rapid detection of infectious pathogens is vital for improving human health. Current detection methods lack the means to detect infectious pathogens in a simple, rapid, and reliable manner at the time and point of need. Functional nucleic acids (FNAs) have the potential to overcome these limitations by acting as key components for point-of-care (POC) biosensors due to their distinctive advantages that include high binding affinities and specificities, excellent chemical stability, ease of synthesis and modification, and compatibility with a variety of signal-amplification and signal-transduction mechanisms.This Account summarizes the work completed in our groups toward developing FNA-based biosensors for detecting bacteria. In vitro selection has led to the isolation of many RNA-cleaving fluorogenic DNAzymes (RFDs) and DNA aptamers that can recognize infectious pathogens, including Escherichia coli, Clostridium difficile, Helicobacter pylori, and Legionella pneumophila. In most cases, a "many-against-many" approach was employed using a DNA library against a crude cellular mixture of an infectious pathogen containing diverse biomarkers as the target to isolate RFDs, with combined counter and positive selections ensuring high specificity toward the desired target. This procedure allows for the isolation of pathogen-specific FNAs without first identifying a suitable biomarker. Multiple target-specific DNA aptamers, including anti-glutamate dehydrogenase (GDH) circular aptamers, anti-degraded toxin B aptamers, and anti-RNase HII aptamers, have also been isolated for the detection of bacteria such as Clostridium difficile. The isolated FNAs have been integrated into fluorescent, colorimetric, and electrochemical biosensors using various signal transduction mechanisms. Both simple-to-use paper-based analytical devices and hand-held electrical devices with integrated FNAs have been developed for POC applications. In addition, signal-amplification strategies, including DNA catenane enabled rolling circle amplification (RCA), DNAzyme feedback RCA, and an all-DNA amplification system using a four-way junction and catalytic hairpin assembly (CHA), have been designed and applied to these systems to further increase their detection sensitivity. The use of these FNA-based biosensors to detect pathogens directly in clinical samples, such as urine, blood, and stool, has now been demonstrated with an outstanding sensitivity of as low as 10 cells per milliliter, highlighting the tremendous potential of using FNA-based sensors in clinical applications. We further describe strategies to overcome the challenges of using FNA-based biosensors in clinical applications, including strategies to improve the stability of FNAs in biological samples and prevent their nonspecific degradation from nucleases and strategies to deal with issues such as signal loss caused by nonspecific binding and biofouling. Finally, the remaining roadblocks for employing FNA-based biosensors in clinical applications are discussed.

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.

Effects of DNA Supercoil Handedness on Catenation by Type II Topoisomerases

Type II topoisomerases interact differently with DNA substrates of opposite supercoil handedness: although the genome is globally unwound, DNA ahead of replication or transcription complexes is overwound, and therefore DNA geometry can influence where the enzyme acts during nucleic acid processes. Human topoisomerase IIa and bacterial topoisomerase IV relax positively supercoiled [(+)SC] DNA faster than negatively supercoiled [(-)SC] molecules, while human topoisomerase IIb shows no preference. An important function of topoisomerase IIa and topoisomerase IV is the decatenation of DNA prior to mitosis. Cellular studies in yeast and bacteria indicate that DNA becomes positively supercoiled through interaction with cellular factors prior to decatenation, but it is not yet known whether the enzyme displays an intrinsic preference for decatenating (+)SC DNA or if these additional cellular factors accelerate the reaction. To determine whether type II topoisomerases possess an intrinsic ability to recognize DNA supercoil handedness during catenation and decatenation, we have carried out a series of in vitro experiments. As these two reactions are in equilibrium, it is important to study the effects of DNA supercoil handedness on both of them. Our studies focused on the effects of (-)SC, (+)SC, and relaxed DNA on catenation by type II topoisomerases. Topoisomerase IIa catenated relaxed DNA ~10-fold faster than (-)SC DNA, and ~45-fold faster than (+)SC DNA. The C-terminal domain of topoisomerase IIa has been previously shown to confer the ability of the enzyme to preferentially relax (+)SC DNA over (-)SC DNA. A deletion construct of topoisomerase IIa that lacked the C-terminal domain showed that this enzyme domain is also required for the preferential catenation of relaxed and (-)SC DNA. Catenation assays with (+)SC substrates did not go to completion, suggesting that chromosomes are less likely to be catenated following positive supercoiling. Additional studies performed with topoisomerase IIa indicated that (-)SC DNA does not relax prior to catenation and, at high concentrations, the enzyme can catenate DNA with very low concentrations of ATP as compared to other strand passage reactions. Topoisomerase IIb displayed slower rates of catenation than topoisomerase IIa and did not discriminate between different supercoil handedness states. In contrast, topoisomerase IV preferentially catenated (+)SC DNA over (-)SC and relaxed DNA. Results show that topoisomerase IIa and topoisomerase IV can distinguish supercoil handedness during catenation and may help determine whether (+)SC decatenation uses a different control mechanism.

Kinetochore-independent mechanisms of sister chromosome separation.

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Chemically modified nucleic acids and DNA intercalators as tools for nanoparticle assembly.

The self-assembly of inorganic nanoparticles to larger structures is of great research interest as it allows the fabrication of novel materials with collective properties correlated to the nanoparticles’ individual characteristics. Recently developed methods for controlling nanoparticle organisation have enabled the fabrication of a range of new materials. Amongst these, the assembly of nanoparticles using DNA has attracted significant attention due to the highly selective recognition between complementary DNA strands, DNA nanostructure versatility, and ease of DNA chemical modification. In this review we discuss the application of various chemical DNA modifications and molecular intercalators as tools for the manipulation of DNA-nanoparticle structures. In detail, we discuss how DNA modifications and small molecule intercalators have been employed in the chemical and photochemical DNA ligation in nanostructures; DNA rotaxanes and catenanes associated with reconfigurable nanoparticle assemblies; and DNA backbone modifications including locked nucleic acids, peptide nucleic acids and borane nucleic acids, which affect the stability of nanostructures in complex environments. We conclude by highlighting the importance of maximising the synergy between the communities of DNA chemistry and nanoparticle self-assembly with the aim to enrich the library of tools available for the manipulation of nanostructures.

Purified Smc5/6 Complex Exhibits DNA Substrate Recognition and Compaction

SummaryEukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.

Regeneration of Burnt Bridges on a DNA Catenane Walker.

DNA walkers are molecular machines that can move with high precision onthe nanoscale due to their structural and functional programmability. Despite recent advances in the field that allow exploring different energy sources, stimuli, and mechanisms of action for these nanomachines, the continuous operation and reusability of DNA walkers remains challenging because in most cases the steps, once taken by the walker, cannot be taken again. Herein we report the path regeneration of a burnt‐bridges DNA catenane walker using RNase A. This walker uses a T7RNA polymerase that produces long RNA transcripts to hybridize to the path and move forward while the RNA remains hybridized to the path and blocks it for an additional walking cycle. We show that RNA degradation triggered by RNase A restores the path and returns the walker to the initial position. RNase inhibition restarts the function of the walker.

Regeneration of Burnt Bridges on a DNA Catenane Walker

AbstractDNA walkers are molecular machines that can move with high precision onthe nanoscale due to their structural and functional programmability. Despite recent advances in the field that allow exploring different energy sources, stimuli, and mechanisms of action for these nanomachines, the continuous operation and reusability of DNA walkers remains challenging because in most cases the steps, once taken by the walker, cannot be taken again. Herein we report the path regeneration of a burnt‐bridges DNA catenane walker using RNase A. This walker uses a T7RNA polymerase that produces long RNA transcripts to hybridize to the path and move forward while the RNA remains hybridized to the path and blocks it for an additional walking cycle. We show that RNA degradation triggered by RNase A restores the path and returns the walker to the initial position. RNase inhibition restarts the function of the walker.

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase–anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.

MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 decatenation checkpoint response. However, the mitotic defects could be accounted for by a minor defect in the activity of Topo II during G2/M. To test this hypothesis, we have tracked at live single cell resolution the dynamics of mitosis in MCPH1 depleted HeLa cells upon catalytic inhibition of Topo II. Our analyses demonstrate that neither chromosome alignment nor segregation are more susceptible to minor perturbation in decatenation in MCPH1 deficient cells, as compared with control cells. Interestingly, MCPH1 depleted cells were more prone to mitotic cell death when decatenation was perturbed. Furthermore, when the G2 arrest that was induced by catalytic inhibition of Topo II was abrogated by Chk1 inhibition, the incidence of mitotic cell death was also increased. Taken together, our data suggest that the MCPH1 lack of function increases mitotic cell hypersensitivity to the catalytic inhibition of Topo II.

Fluorescent aptasensors for parallel analysis of biomolecules based on interlocked DNA catenane nanomachines

Stimuli-responsive DNA catenane nanomachines have received considerable interest in the area of DNA nanotechnology. However, the sensing and bio-sensing applications of DNA catenane nanomachines are rarely explored. Herein, the biological small molecule and protein responsive DNA catenane nanomachines were designed by utilizing the specific aptamer/target interaction. In the presence of the target, the blocked catalytically inactive DNA catenane can be activated, while generating the metal ion-dependent DNAzyme as the signal output unit. The method allows the fluorescent detection of ATP and lysozyme with a detection limit of 20 nM and 200 pM, respectively. More importantly, through the use of different fluorophores as labels, the parallel analysis of ATP and lysozyme in the mixed solution was demonstrated based on two interlocked DNA catenanes. Compared with linear scaffold, the interlocked DNA rings showed enhanced stability against enzyme degradation and easily realized intramolecular reconfiguration. Due to its inherent advantages, the interlocked DNA catenane holds great promise in the field of DNA based sensors and nanodevices.

Facile Characterization of Topology of DNA Catenanes

During the preparation of single-stranded DNA catenanes, topological isomers of different linking numbers (Lk) are intrinsically produced, and they must be separated from each other to construct sophisticated nanostructures accurately. In many previous studies, however, mixtures of these isomers were directly employed to construct nanostructures without sufficient characterization. Here, we present a method that easily and clearly characterizes the isomers by polyacrylamide gel electrophoresis. To the mixtures of topological isomers of [2]catenanes, two-strut oligonucleotides, which are complementary with a part of both rings, were added to connect the rings and fix the whole conformations of isomers. As a result, the order of migration rate was always Lk3 > Lk2 > Lk1, irrespective of gel concentration. Thus, all the topological isomers were unanimously characterized by only one polyacrylamide gel electrophoresis experiment. Well-characterized DNA catenanes are obtainable by this two-strut strategy, opening the way to more advanced nanotechnology.

Checkpoint inhibition of origin firing prevents DNA topological stress.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes.

Due to helical structure of DNA, massive amounts of positive supercoils are constantly introduced ahead of each replication fork. Positive supercoiling inhibits progression of replication forks but various mechanisms evolved that permit very efficient relaxation of that positive supercoiling. Some of these mechanisms lead to interesting topological situations where DNA supercoiling, catenation and knotting coexist and influence each other in DNA molecules being replicated. Here, we first review fundamental aspects of DNA supercoiling, catenation and knotting when these qualitatively different topological states do not coexist in the same circular DNA but also when they are present at the same time in replicating DNA molecules. We also review differences between eukaryotic and prokaryotic cellular strategies that permit relaxation of positive supercoiling arising ahead of the replication forks. We end our review by discussing very recent studies giving a long-sought answer to the question of how slow DNA topoisomerases capable of relaxing just a few positive supercoils per second can counteract the introduction of hundreds of positive supercoils per second ahead of advancing replication forks.