DNA Condensation Research Articles

Enzyme-Responsive DNA Condensates.

Membrane-less compartments and organelles are widely acknowledged for their role in regulating cellular processes, and there is an urgent need to harness their full potential as both structural and functional elements of synthetic cells. Despite rapid progress, synthetically recapitulating the nonequilibrium, spatially distributed responses of natural membrane-less organelles remains elusive. Here, we demonstrate that the activity of nucleic-acid cleaving enzymes can be localized within DNA-based membrane-less compartments by sequestering the respective DNA or RNA substrates. Reaction-diffusion processes lead to complex nonequilibrium patterns, dependent on enzyme concentration. By arresting similar dynamic patterns, we spatially organize different substrates in concentric subcompartments, which can be then selectively addressed by different enzymes, demonstrating spatial distribution of enzymatic activity. Besides expanding our ability to engineer advanced biomimetic functions in synthetic membrane-less organelles, our results may facilitate the deployment of DNA-based condensates as microbioreactors or platforms for the detection and quantitation of enzymes and nucleic acids.

A Novel Regulatory Motif at the Hinge Dimer Interface of the MksB Mediates Dimerization and DNA Binding Activity.

The Structural Maintenance of Chromosome (SMC) protein is essential for various cellular processes, including chromosome organization, DNA repair, and genome stability. MksB, an alternative SMC protein present in prokaryotes, comprises a hinge dimerization domain and an ABC ATPase head domain linked by a coiled-coil arm. While hinge dimerization in bacterial and eukaryotic SMCs is attributed to conserved glycines, our study unveils the critical role of a novel KDDR motif located at a loop near the hinge dimer interface in Mycobacterium smegmatis MksB (MsMksB). We demonstrate the regulatory role of this motif in MsMksB dimerization and DNA binding activity. The K600D mutation in the KDDR motif induces MsMksB dimer-to-monomer conversion, highlighting the significance of this motif in MsMksB dimerization. Mass spectrometry-based mapping of the DNA binding site revealed the lysine's involvement in protein-DNA interaction. Monomers of the hinge domain lose DNA binding activity, and MsMksB single-arm mutants exhibit reduced DNA binding and ATPase activity, underscoring the importance of hinge-mediated dimerization in MsMksB function. Notably, the R603D mutant retains dimerization but shows compromised ATPase and DNA binding activities. Mutants with defective ATPase activity exhibit impaired DNA condensation in vivo. These findings provide novel regulatory insight into the mechanism of MksB dimerization and DNA binding, uncovering the fundamental processes of chromosome condensation and segregation.

Antioxidant, cytotoxic, anti-migratory, and pro-apoptotic effects of Bolanthus turcicus extracts on head and neck cancer cells.

Investigation of various plant extracts using in-vitro/in-vivo assays has emerged as a promising avenue for identifying potential pharmacophores that can be developed into therapeutic drugs. This study aims to assess the bioactive compounds and antioxidant capacity of the Bolanthus turcicus (B. turcicus) and to investigate the effects on head and neck cancer (HNC) cell lines. Methanol (MeOH), ethyl acetate (EA) and aqueous (Aq) extracts were prepared from B. turcicus, and the amount of total phenolic content (TPC) and total flavonoid content (TFC) in the extracts were analyzed by the Folin-Ciocalteu and Aluminum chloride method, respectively. In addition, the total antioxidant capacity and iron reducing potential of B. turcicus extracts were determined by the Phosphomolybdenum and Ferric ion reducing antioxidant power (FRAP) method. The effect of B. turcicus on HEp-2, SCC-90, SCC-9, FaDu HNC cell viability, motility, and cell-nuclear morphology was evaluated by MTT, scratch-wound healing assay, and Pllalloidin-DAPI staining, respectively. The effect of B. turcicus on the expression of CASP-3, BAX, and BCL-2 genes at the mRNA, protein, and intracellular level was evaluated by quantitative PCR (qPCR), western blot, and immunofluorescence staining. Moreover, Annexin V-FITC/PI, was used in flow cytometry to investigate the effect of B. turcicus on apoptosis. The MeOH extract exhibited the highest phenolic content, flavonoid content and antioxidant activity (p < 0.05 for all). HNC cells treated with extracts indicated delayed wound healing and decreased motility (p < 0.05 for all). Analysis of annexin V-PI staining indicated that the B. turcicus extracts induced apoptosis but not viability and necrosis in the HNC cell (p < 0.05 for all). Moreover, qPCR data regarding the apoptotic mechanism showed that the extracts could induce apoptosis by upregulation of pro-apoptotic CASP-3 and BAX genes and downregulation of anti-apoptotic BCL-2 gene (p < 0.05 for all). The expression of protein and intracellular levels of CASP-3 and BAX were increased, while the BCL-2 was decreased in cells treated with the extracts (p < 0.05 for all). In addition, diffuse pycnosis and DNA condensation in HNC cell nuclei, confirming apoptotic cell death (p < 0.05 for all). This study data indicated that B. turcicus extracts have antioxidant, cytotoxic, anti-migratory and pro-apoptotic activity. In conclusion, it has been shown that B. turcicus can be used as a potential therapeutic agent against HNC.

DNA supercoiling enhances DNA condensation by ParB proteins.

The ParABS system plays a critical role in bacterial chromosome segregation. The key component of this system, ParB, loads and spreads along DNA to form a local protein-DNA condensate known as a partition complex. As bacterial chromosomes are heavily supercoiled due to the continuous action of RNA polymerases, topoisomerases and nucleoid-associated proteins, it is important to study the impact of DNA supercoiling on the ParB-DNA partition complex formation. Here, we use an in-vitro single-molecule assay to visualize ParB on supercoiled DNA. Unlike most DNA-binding proteins, individual ParB proteins are found to not pin plectonemes on supercoiled DNA, but freely diffuse along supercoiled DNA. We find that DNA supercoiling enhances ParB-DNA condensation, which initiates at lower ParB concentrations than on DNA that is torsionally relaxed. ParB proteins induce a DNA-protein condensate that strikingly absorbs all supercoiling writhe. Our findings provide mechanistic insights that have important implications for our understanding of bacterial chromosome organization and segregation.

Metal Ion-Condensed DNA Nanoparticle Library: Phase Separation and Transition and Antisense Therapy Applications.

DNA condensation has long been investigated as a fundamental cellular activity and is known to be driven by the mediation of diverse condensing agents. The phase behaviors of DNA during condensation are particularly interesting because the complicated molecular structure of natural nucleotides fundamentally allows electrostatic, coordinate covalent, and various other secondary interactions with the condensing agents. Recently, metal ion (Mn+)-induced DNA condensation has emerged as a powerful approach to synthesizing nanoparticulate DNA structures suitable for therapeutic gene delivery. However, how the DNA phase changes during Mn+-induced DNA condensation has rarely been observed and is not understood yet. In this study, a library of Mn+-condensed DNA nanoparticles (Mn+-CDNPs) was established using 30 different types of Mn+s, and their phase behaviors during condensation were elucidated using spherical nucleic acids (SNAs) as electron microscopic labels. Importantly, the phase transition and separation of DNA were demonstrated to be driven by the Mn+s into either the growth of individual DNA particles or the fission of bulky DNA aggregates. Pt2+ and Eu3+ were chosen as model systems for the demonstration. The hard and soft acid nature of Mn+ is presumably the underlying driving force of these phase transitions. In addition, the Mn+-controlled anticancer therapeutic efficiency of the Mn+-CDNP library as a state-of-the-art gene delivery platform was demonstrated even for unmodified antisense oligonucleotides in association with the potential toxicity of the Mn+s released from the Mn+-CDNPs. This comprehensive study of the Mn+-dependent condensation of nucleic acids provides profound insights into the chemistry of the nucleic acid-Mn+ interactions and the reliable theragnostic applications of Mn+-CDNPs as functional nucleic acid nanostructures.

Converting Multiple- to Single-DNA-Tethered Beads and Removing Only-One-End-Tethered DNA in High-Throughput Stretching.

S-DNA is a double-stranded DNA that forms under tensions of >65 pN. Here, we report that S-DNA resists the cleavage of Cas12a and the restriction endonuclease SmaI. Taking advantage of this resistance, in magnetic tweezer experiments, we developed an assay to convert multiple-DNA-tethered beads into single-DNA-tethered beads and remove the only-one-end-tethered DNA molecule by cleaving the DNA that does not transition to S-DNA at about 80 pN. When multiple DNA molecules are tethered to a single bead, they share the tension, exist in the B-form, and allow the cleavage. Only-one-end-tethered DNA molecules, free of tension, are also cleaved. In versatile types of experiments, we proved the broad applications of this assay: measuring the correct DNA elasticity and DNA condensation dynamics by avoiding the false results due to interference of only-one-end-tethered DNA molecules and quantifying the accurate cleavage rates of Cas12a and the restriction endonucleases by eliminating the error caused by multiple-DNA-tethered beads. This convenient assay ensures correct and accurate results in high-throughput DNA stretching experiments.

DNA-Origami-Armored DNA Condensates.

DNA condensates, formed by liquid-liquid phase separation (LLPS), emerge as promising soft matter assemblies for creating artificial cells. The advantages of DNA condensates are their molecular permeability through the surface due to their membrane-less structure and their fluidic property. However, they face challenges in the design of their surface, e. g., unintended fusion and less regulation of permeable molecules. Addressing them, we report surface modification of DNA condensates with DNA origami nanoparticles, employing a Pickering-emulsion strategy. We successfully constructed core-shell structures with DNA origami coatings on DNA condensates and further enhanced the condensate stability toward fusion via connecting DNA origamis by responding to DNA input strands. The 'armoring' prevented the fusion of DNA condensates, enabling the formation of multicellular-like structures of DNA condensates. Moreover, the permeability was altered through the state change from coating to armoring the DNA condensates. The armored DNA condensates have significant potential for constructing artificial cells, offering increased surface stability and selective permeability for small molecules while maintaining compartmentalized space and multicellular organization.

Nanoscale imaging of DNA-RNA identifies transcriptional plasticity at heterochromatin.

The three-dimensional structure of DNA is a biophysical determinant of transcription. The density of chromatin condensation is one determinant of transcriptional output. Chromatin condensation is generally viewed as enforcing transcriptional suppression, and therefore, transcriptional output should be inversely proportional to DNA compaction. We coupled stable isotope tracers with multi-isotope imaging mass spectrometry to quantify and image nanovolumetric relationships between DNA density and newly made RNA within individual nuclei. Proliferative cell lines and cycling cells in the murine small intestine unexpectedly demonstrated no consistent relationship between DNA density and newly made RNA, even though localized examples of this phenomenon were detected at nuclear-cytoplasmic transitions. In contrast, non-dividing hepatocytes demonstrated global reduction in newly made RNA and an inverse relationship between DNA density and transcription, driven by DNA condensates at the nuclear periphery devoid of newly made RNA. Collectively, these data support an evolving model of transcriptional plasticity that extends at least to a subset of chromatin at the extreme of condensation as expected of heterochromatin.

Implicit Solvent with Explicit Ions Generalized Born Model in Molecular Dynamics: Application to DNA.

The ion atmosphere surrounding highly charged biomolecules, such as nucleic acids, is crucial for their dynamics, structure, and interactions. Here, we develop an approach for the explicit treatment of ions within an implicit solvent framework suitable for atomistic simulations of biomolecules. The proposed implicit solvent/explicit ions model, GBION, is based on a modified generalized Born (GB) model; it includes separate, modified GB terms for solute-ion and ion-ion interactions. The model is implemented in the AMBER package (version 24), and its performance is thoroughly investigated in atomistic molecular dynamics (MD) simulations of double-stranded DNA on a microsecond time scale. The aggregate characteristics of monovalent (Na+ and K+) and trivalent (Cobalt Hexammine, CoHex3+) counterion distributions around double-stranded DNA predicted by the model are in reasonable agreement with the experiment (where available), all-atom explicit water MD simulations, and the expectation from the Manning condensation theory. The radial distributions of monovalent cations around DNA are reasonably close to the ones obtained using the explicit water model: expressed in units of energy, the maximum deviations of local ion concentrations from the explicit solvent reference are within 1 kBT, comparable to the corresponding deviations expected between different established explicit water models. The proposed GBION model is able to simulate DNA fragments in a large volume of solvent with explicit ions with little additional computational overhead compared with the fully implicit GB treatment of ions. Ions simulated using the developed model explore conformational space at least 2 orders of magnitude faster than in the explicit solvent. These advantages allowed us to observe and explore an unexpected "stacking" mode of DNA condensation in the presence of trivalent counterions (CoHex3+) that was revealed by recent experiments.

Traditionally used edible medicinal plants protect against rotenone induced toxicity in SH-SY5Y cells-a prospect for the development of herbal nutraceuticals

Plants are good sources of pharmacologically active compounds. The present study aimed to examine the neuroprotective potentials of the methanol extracts of Salix tetrasperma Roxb. leaf (STME) and Plantago asiatica L. (PAME), two edibles medicinal plants of Manipur, India against neurotoxicity induced by rotenone in SH-SY5Y cells. Free radical quenching activities were evaluated by ABTS and DPPH assays. The cytotoxicity of rotenone and the neuronal survival were assessed by MTT assay and MAP2 expression analysis. DCF-DA, Rhodamine 123 (Rh-123), and DAPI measured the intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and apoptotic nuclei, respectively. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were also assessed. LC-QTOF-MS analysis was performed for the identification of the compounds present in STME and PAME. The study showed that both the plant extracts (STME and PAME) showed antioxidant and neuroprotective capabilities in rotenone-induced neurotoxicity by preventing oxidative stress through the reduction of intracellular ROS levels and reversing the activities of GPx, SOD, and CAT caused by rotenone. Further, both plants prevented apoptotic cell death by normalizing the steady state of MMP and protecting nuclear DNA condensation. LC-QTOF-MS analysis shows the presence of known neuroprotective compounds like uridine and gabapentin in STME and PAME respectively. The two plants might be an important source of natural antioxidants and nutraceuticals with neuroprotective abilities. This could be investigated further to formulate herbal nutraceuticals for the treatment of neurodegenerative disease like Parkinson's disease.

Interfacial Polymerization Fabricated Polydopamine Capsules as a Step Toward a Photothermal Protocell Model

AbstractPhotothermal is a significant solar energy conversion process on Earth, and photothermal‐responsive protocells are a potential model for prebiotic chemistry. Here, polydopamine microcapsules are fabricated via emulsion droplets‐templated interfacial polymerization and investigated their potential as synthetic protocell models for photothermal conversion and utilization. DNAzyme‐mediated dopamine peroxidation and polymerization occur at the water/oil interface of the emulsion droplets, facilitated by a biphasic microfluidic technique, leading to the formation of uniform polydopamine microcapsules, which retain structural integrity upon transfer into a continuous water phase. These microcapsules exhibit size‐dependent semi‐permeability and high photothermal capability due to their broad spectral absorbance. The photothermal‐mediated temperature rise within the microcapsules triggers DNA denaturation and rearrangement, and intermittent photothermal irradiation modulates reversible phase separation of DNA condensates, mimicking membrane‐less organelles. This work suggests that interfacial polymerization fabricated polydopamine capsules are a plausible photothermal protocell model that can demonstrate aspects of primitive abiotic cellularity.

PEITC Induces DNA Damage and Inhibits DNA Repair-Associated Proteins in Human Retinoblastoma Cells In Vitro.

Phenethyl isothiocyanate (PEITC), a natural product, exists in biological activities, including anticancer activity in many human cancer cells. No information shows that PEITC affects DNA damage in human retinoblastoma (RB) cells in vitro. In this study, the aim of experiments was to determine whether PEITC decreased total viable cell number or not by inducing protein expressions involved in DNA damage and repair in Y79 RB cells in vitro. Total cell viability was measured by PI exclusion assay, and PEITC reduced the total Y79 viable cell numbers in a dose-dependent manner. DNA condensation and DNA impairment were conducted by DAPI staining and comet assays, respectively, in Y79 cells. The findings show that PEITC induced DNA condensation dose-dependently based on the brighter fluorescence of cell nuclei stained by DAPI staining. PEITC-induced DNA damage showed a more extended DNA migration smears than that of the control, which was performed by a comet assay. Western blotting was performed to measure the protein expressions involved in DNA damage and repair, which showed that PEITC at 2.5-10 μM increased NRF2, HO-1, SOD (Mn), and catalase; however, it decreased SOD (Cu/Zn) except 10 μM PEITC treatment, and decreased glutathione, which were associated with oxidativestress. Furthermore, PEITC increased DNA-PK, MDC1, H2A.XpSer139, ATMpSer1981, p53, p53pSer15, PARP, HSP70, and HSP90, but decreased TOPIIα, TOPIIβ, and MDM2pSer166 that were associated with DNA damage and repair mechanism in Y79 cells. The examination from confocal laser microscopy shows that PEITC increased H2A.XpSer139 and p53pSer15, and decreased glutathione and TOPIIα in Y79 cells. In conclusion, the cytotoxic effects of PEITC on reducing the number of viable cells may be due to the induction of DNA damage and the alteration of DNA repair proteins in Y79 cells in vitro.

Fractionated Leaf Extracts of Ocimum gratissimum Inhibit the Proliferation and Induce Apoptosis of A549 Lung Adenocarcinoma Cells.

Previous in vitro studies in our laboratory demonstrated that ethyl acetate (P2) and water- soluble (PS/PT1) fractionated leaf extracts of Ocimum gratissimum inhibit the proliferation of prostate cancer cells. It has been reported that the crude aqueous extract induces apoptosis in lung adenocarcinoma cells; however, the efficacy of the fractionated extracts against these cells remains unclear. In the present study, we hypothesized that the ability of the fractionated extracts to inhibit proliferation and induce apoptosis is associated with the activation of pro-apoptotic proteins and induction of DNA condensation in A549 cells. Ocimum gratissimum was cultivated and its leaves were harvested, extracted, and fractionated to produce fractions P2 and PS/PT1. Anti-proliferative activity was assessed by direct cell count. For morphological characterization of apoptosis, 4',6-diamidino-2-phenylindole staining was employed. Western blot analysis was performed to evaluate the apoptotic activity of the fractionated extracts. In data generated from anti-proliferation studies, P2 significantly inhibited cell proliferation in a concentration-dependent manner; PS/PT1 elicited a decrease in the viability of cells, occurring at 500 µg/mL. 4',6-diamidino-2-phenylindole staining revealed the induction of apoptosis, as evidenced by the formation of apoptotic bodies. Increased levels of pro-apoptotic proteins were observed as the concentrations of the fractionated extracts increased. These results suggest that fractionated leaf extracts of Ocimum gratissimum inhibit the proliferation and induce apoptosis of A549 cells.

Fdo1, Fkh1, Fkh2, and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae.

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

Zafirlukast induces DNA condensation and has bactericidal effect on replicating Mycobacterium abscessus.

Mycobacterium abscessus infections are emerging in cystic fibrosis patients, and treatment success rate in these patients is only 33% due to extreme antibiotic resistance. Thus, new treatment options are essential. An interesting target could be Lsr2, a nucleoid-associated protein involved in mycobacterial virulence. Zafirlukast is a Food and Drug Administration (FDA)-approved drug against asthma that was shown to bind Lsr2. In this study, zafirlukast treatment is shown to reduce M. abscessus growth, with a minimal inhibitory concentration of 16 µM and a bactericidal concentration of 64 µM in replicating bacteria only. As an initial response, DNA condensation, a known stress response of mycobacteria, occurs after 1 h of treatment with zafirlukast. During continued zafirlukast treatment, the morphology of the bacteria alters and the structural integrity of the bacteria is lost. After 4 days of treatment, reduced viability is measured in different culture media, and growth of M. abscessus is reduced in a dose-dependent manner. Using transmission electron microscopy, we demonstrated that the hydrophobic multilayered cell wall and periplasm are disorganized and ribosomes are reduced in size and relocalized. In summary, our data demonstrate that zafirlukast alters the morphology of M. abscessus and is bactericidal at 64 µM. The bactericidal concentration of zafirlukast is relatively high, and it is only effective on replicating bacteria but as zafirlukast is an FDA-approved drug, and currently used as an anti-asthma treatment, it could be an interesting drug to further study in in vivo experiments to determine whether it could be used as an antibiotic for M. abscessus infections.

Mechanism of Cationic Lipid Induced DNA Condensation: Lipid-DNA Coordination and Divalent Cation Charge Fluctuations.

The condensation of nucleic acids by lipids is a widespread phenomenon in biology with crucial implications for drug delivery. However, the mechanisms of DNA assembly in lipid bilayers remain insufficiently understood due to challenges in measuring and assessing each component's contribution in the lipid-DNA-cation system. This study uses all-atom molecular dynamics simulations to investigate DNA condensation in cationic lipid bilayers. Our exhaustive exploration of the thermodynamic factors reveals unique roles for phospholipid head groups and cations. We observed that bridging cations between lipid and DNA drastically reduce charges, while mobile magnesium cations "ping-ponging" between double strands create charge fluctuations. While the first factor stabilizes the DNA-lipid complex, the latter creates attractive forces to induce the spontaneous condensation of DNAs. This novel mechanism not only sheds light on the current data regarding cationic lipid-induced DNA condensation but also provides potential design strategies for creating efficient gene delivery vectors for drug delivery.

Gene therapy by virus-like self-spooling toroidal DNA condensates for revascularization of hindlimb ischemia

Peripheral arterial diseases (PAD) have been reported to be the leading cause for limb amputations, and the current therapeutic strategies including antiplatelet medication or intervene surgery are reported to not clinically benefit the patients with high-grade PAD. To this respect, revascularization based on angiogenetic vascular endothelial growth factor (VEGF) gene therapy was attempted for the potential treatment of critical PAD. Aiming for transcellular delivery of VEGF-encoding plasmid DNA (pDNA), we proposed to elaborate intriguing virus-like DNA condensates, wherein the supercoiled rigid micrometer-scaled plasmid DNA (pDNA) could be regulated in an orderly fashion into well-defined nano-toroids by following a self-spooling process with the aid of cationic block copolymer poly(ethylene glycol)-polylysine at an extraordinary ionic strength (NaCl: 600 mM). Moreover, reversible disulfide crosslinking was proposed between the polylysine segments with the aim of stabilizing these intriguing toroidal condensates. Pertaining to the critical hindlimb ischemia, our proposed toroidal VEGF-encoding pDNA condensates demonstrated high levels of VEGF expression at the dosage sites, which consequently contributed to the neo-vasculature (the particularly abundant formation of micro-vessels in the injected hindlimb), preventing the hindlimb ischemia from causing necrosis at the extremities. Moreover, excellent safety profiles have been demonstrated by our proposed toroidal condensates, as opposed to the apparent immunogenicity of the naked pDNA. Hence, our proposed virus-like DNA condensates herald potentials as gene therapy platform in persistent expressions of the therapeutic proteins, and might consequently be highlighted in the management of a variety of intractable diseases.

Endonucleosis mediates internalization of cytoplasm into the nucleus

Setd8 regulates transcription elongation, mitotic DNA condensation, DNA damage response and replication licensing. Here we show that, in mitogen-stimulated liver-specific Setd8-KO mice, most of the hepatocytes are eliminated by necrosis but a significant number of them survive via entering a stage exhibiting several senescence-related features. Setd8-deficient hepatocytes had enlarged nuclei, chromosomal hyperploidy and nuclear engulfments progressing to the formation of intranuclear vesicles surrounded by nuclear lamina. These vesicles contain glycogen, cytoplasmic proteins and even entire organelles. We term this process “endonucleosis”. Intranuclear vesicles are absent in hepatocytes of Setd8/Atg5 knockout mice, suggesting that the process requires the function of the canonical autophagy machinery. Endonucleosis and hyperploidization are temporary, early events in the surviving Setd8-deficient cells. Larger vesicles break down into microvesicles over time and are eventually eliminated. The results reveal sequential events in cells with extensive DNA damage, which function as part of survival mechanisms to prevent necrotic death.

Phase-separated ParB enforces diverse DNA compaction modes and stabilizes the parS-centered partition complex.

The tripartite ParABS system mediates chromosome segregation in the majority of bacterial species. Typically, DNA-bound ParB proteins around the parS sites condense the chromosomal DNA into a higher-order multimeric nucleoprotein complex for the ParA-driven partition. Despite extensive studies, the molecular mechanism underlying the dynamic assembly of the partition complex remains unclear. Herein, we demonstrate that Bacillus subtilis ParB (Spo0J), through the multimerization of its N-terminal domain, forms phase-separated condensates along a single DNA molecule, leading to the concurrent organization of DNA into a compact structure. Specifically, in addition to the co-condensation of ParB dimers with DNA, the engagement of well-established ParB condensates with DNA allows for the compression of adjacent DNA and the looping of distant DNA. Notably, the presence of CTP promotes the formation of condensates by a low amount of ParB at parS sites, triggering two-step DNA condensation. Remarkably, parS-centered ParB-DNA co-condensate constitutes a robust nucleoprotein architecture capable of withstanding disruptive forces of tens of piconewton. Overall, our findings unveil diverse modes of DNA compaction enabled by phase-separated ParB and offer new insights into the dynamic assembly and maintenance of the bacterial partition complex.

Water structural effects on DNA–DNA interactions and homologous recognition

Comprehending the principles underlying the interaction between DNA molecules in electrolyte solution is of crucial importance for understanding the phenomena of DNA condensation, recognition of homologous genes, and properties of DNA liquid crystals. Although DNA was considered in all its helical complexity over the last two decades, only a primitive, local, electrostatic model of the aqueous solution was considered. The forces acting between DNA molecules however must be influenced by the structure of the liquid separating them. In this paper we make a step towards unravelling earlier neglected effects of water structure on DNA–DNA interactions. We develop a model of DNA interaction accounting for the nonlocal dielectric response of water and of the electrolyte plasma dissolved in it. We focus particularly on the study of the dipolar overscreening effect, which leads to decaying oscillations of the electrostatic potential about DNA, and how it will affect electrostatic energy surfaces as a function of interaxial separation and mutual azimuthal orientation of the parallelly aligned interacting DNAs. We found that going beyond the classical electrostatic description of water (the so-called primitive model) reveals a complex oscillatory interaction surface in space, with many possible metastable configurations. These however vanish rapidly with smearing of the DNA charge distribution, due to dephasing of the oscillatory components of the interaction. This dephasing results in the suppression of oscillation amplitudes and leads to the dominance of non-oscillatory components in spatial dipolar correlations and the Debye screening. These dominant correlation patterns are shown to lead to a substantial enhancement of the difference between the interaction of homologous and nonhomologous DNA sequences, deepening and sharpening the homology recognition well.