Superhelical DNA Research Articles

Synthesis and DNA Cleavage Activity of Cationic Methylene-Blue-Backboned Polymers.

Methods of DNA cleavage have broad bioapplications in gene editing, disease treatment, and biosensor design. The traditional method for DNA cleavage is mainly through oxidation or hydrolysis mediated by small molecules or transition metal complexes. However, DNA cleavage by artificial nucleases using organic polymers has been rarely reported. Methylene blue has been extensively studied in the fields of biomedicine and biosensing due to its excellent singlet oxygen yield, redox properties, and good DNA affinity. Methylene blue mainly relies on light and oxygen for DNA cleavage, and the cutting rate is slow. Here, we synthesize cationic methylene-blue-backboned polymers (MBPs) that can bind DNA efficiently and induce DNA cleavage through free radical mechanisms in the absence of light and exogenous reagents, showing high-efficiency nuclease activity. In addition, MBPs with different structures showed selectivity for DNA cleavage, and the cleavage efficiency of the flexible structure was significantly higher than that of the rigid structure. Studies on the DNA cleavage mechanism have shown that the cleavage mechanism of MBPs is not through the common ROS-mediated oxidative cleavage pathway, but through the radical of MBP• inducing DNA cleavage. Meanwhile, MBPs can simulate topoisomerase I (Topo I)-mediated topological rearrangement of superhelical DNA. This work paved a way for the application of MBPs in the field of artificial nucleases.

Interaction of C-terminal p53 isoforms depends strongly upon DNA sequence and topology

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and β isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53β and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

Models of topological barriers and molecular motors of bacterial DNA

ABSTRACT Bacterial genomes are partitioned into kilobases long domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbours. This is made possible by proteins like the LacI repressor, which behave like topological barriers and block the diffusion of torsion along the DNA. Other proteins, like DNA gyrases and RNA polymerases, called molecular motors, use the energy released by the hydrolysis of ATP to apply forces and/or torques to the DNA and modify its superhelicity. Here, we report on simulation work aimed at enlightening the interplay between DNA supercoiling, topological barriers, and molecular motors. To this end, we developed a coarse-grained Hamiltonian model of topological barriers and a model of molecular motors and investigated their properties through Brownian dynamics simulations. We discuss their influence on the contact map of a model nucleoid and the steady state values of twist and writhe in the DNA. These coarse-grained models, which are able to predict the dynamics of plectonemes depending on the position of topological barriers and molecular motors, should prove helpful to back up experimental efforts, like the development of Chromosome Conformation Capture techniques, and decipher the organisational mechanisms of bacterial chromosomes.

Sex-specific variation in R-loop formation in Drosophila melanogaster.

R-loops are three-stranded nucleotide structures consisting of a DNA:RNA hybrid and a displaced ssDNA non-template strand. Previous work suggests that R-loop formation is primarily determined by the thermodynamics of DNA:RNA binding, which are governed by base composition (e.g., GC skew) and transcription-induced DNA superhelicity. However, R-loops have been described at genomic locations that lack these properties, suggesting that they may serve other context-specific roles. To better understand the genetic determinants of R-loop formation, we have characterized the Drosophila melanogaster R-loop landscape across strains and between sexes using DNA:RNA immunoprecipitation followed by high-throughput sequencing (DRIP-seq). We find that R-loops are associated with sequence motifs that are G-rich or exhibit G/C skew, as well as highly expressed genes, tRNAs, and small nuclear RNAs, consistent with a role for DNA sequence and torsion in R-loop specification. However, we also find motifs associated with R-loops that are A/T-rich and lack G/C skew as well as a subset of R-loops that are enriched in polycomb-repressed chromatin. Differential enrichment analysis reveals a small number of sex-biased R-loops: while non-differentially enriched and male-enriched R-loops form at similar genetic features and chromatin states and contain similar sequence motifs, female-enriched R-loops form at unique genetic features, chromatin states, and sequence motifs and are associated with genes that show ovary-biased expression. Male-enriched R-loops are most abundant on the dosage-compensated X chromosome, where R-loops appear stronger compared to autosomal R-loops. R-loop-containing genes on the X chromosome are dosage-compensated yet show lower MOF binding and reduced H4K16ac compared to R-loop-absent genes, suggesting that H4K16ac or MOF may attenuate R-loop formation. Collectively, these results suggest that R-loop formation in vivo is not fully explained by DNA sequence and topology and raise the possibility that a distinct subset of these hybrid structures plays an important role in the establishment and maintenance of epigenetic differences between sexes.

Characterization of [Ru(bpy)2(diamine)]2+ complexes and their DNA binding and cleavage, BSA interaction, cytotoxic, and anticancer mechanistic properties

The ruthenium(II) complexes of the type [Ru(bpy)2(diamine)](PF6)21–3, where diamine is ethylenediamine (en, 1) or N,N-dimethylethylenediamine (dmen, 2) and N,N-diethyl-ethylenediamine (deen, 3) and bpy is 2,2′-bipyridine has been isolated and characterized. The X-ray crystal structures of 1 and 2 reveal that the compounds possess a distorted octahedral geometry composed of two amine nitrogen atoms of the diamine ligand (1, en; 2, dmen) and four nitrogen atoms of the two bpy ligands, which selectively forms a “cis-α” (2) or “cis-β” (1) isomer. The 1H and 13C NMR spectral data of the complexes disclose the nature of metal-ligand bonding, conformations of the chelate rings, and absolute configurations, which indicate that the two bpy ligands maintain their tendency to form geometrical isomers even in solution. They exhibit dπ → π* MLCT transitions, show a ligand-based radical anion (3MLCT) luminescence, and display one quasi-reversible and one non-reversible metal-based oxidative process corresponding to RuII/RuIII and RuIII/RIV redox couples respectively. They interacted with calf thymus (CT) DNA and the mode of interaction was found to be groove binding, as determined by various spectral and redox techniques. Upon interaction with CT DNA 1–3 show a decrease in current and less positive shift in the formal potential in the differential pulse voltammograms suggesting that they are involved in electrocatalytic guanine oxidation. Interestingly, 1–3 alters the DNA superhelicity upon binding with supercoiled pUC19 plasmid DNA with a reductant. Further, the quenching of the tryptophan emission of BSA in the presence of 1–3 is static. The binding is mainly entropy-driven and hydrophobic forces played a major role. The DNA and protein binding affinity and the DNA cleavage activity is in the order 2 > 3 > 1, and the higher DNA and protein binding affinity and DNA cleavage activity of 2 and 3 illustrates the strong involvement of the methyl (dmen) and ethyl (deen) groups in the hydrophobic interaction with the DNA and protein. Furthermore, the ruthenium(II) complexes, 2 and 3 exhibit moderate cytotoxicity against human breast adenocarcinoma MCF-7 and human cervical carcinoma HeLa (IC50: 48 h, 28–45 μM) cells, whereas 2 displays the best activity (IC50: 48 h, 28 μM) for MCF-7 cancer cells, which is better than that of cisplatin. Both the complexes displayed excellent selectivity by being relatively inactive against the Madin-Darby bovine kidney (MDBK) normal cells with selectivity index (SI) values ranging from 5.6 to 8.9. They block the cell cycle progression of MCF-7 cells in the G1 (2) and S (3) phases. FACSVerse analyses are suggestive of reactive oxygen species (ROS) generation and apoptotic cell death induced by 2 and 3. The induction of apoptosis in MCF-7 cells was shown by Annexin V with propidium iodide (PI) and 4’,6-diamidino-2-phenylindole (DAPI) staining methods.

Molecular Characterization of a New Tetrodotoxin-Binding Protein, Peroxiredoxin-1, from Takifugu bimaculatus.

Pufferfish are considered a culinary delicacy but require careful preparation to avoid ingestion of the highly toxic tetrodotoxin (TTX), which accumulates in certain tissues. In this study, the tissue distribution of peroxiredoxin-1 from Takifugu bimaculatus was investigated. The peroxiredoxin-1 protein was obtained by in vitro recombinant expression and purification. The recombinant protein had a strong ability to scavenge hydroxyl radicals, protect superhelical DNA plasmids from oxidative damage, and protect L929 cells from H2O2 toxicity through in vitro antioxidant activity. In addition, we verified its ability to bind to tetrodotoxin using surface plasmon resonance techniques. Further, recombinant proteins were found to facilitate the entry of tetrodotoxin into cells. Through these analyses, we identified, for the first time, peroxiredoxin-1 protein from Takifugu bimaculatus as a potential novel tetrodotoxin-binding protein. Our findings provide a basis for further exploration of the application of peroxiredoxin-1 protein and the molecular mechanisms of tetrodotoxin enrichment in pufferfish.

Superhelical DNA liquid crystals from dendrimer-induced DNA compaction.

Electrostatic compaction of double stranded DNA induced by a positively charged poly(amidoamine) (PAMAM) dendrimer of generation four (G4) was found to produce two unique types of DNA mesophases, in which the DNA bent into superhelices packed in a tetragonal or hexagonal lattice. The structure formed at a lower dendrimer charge density was three-dimensionally (3D) ordered, as characterized by the P41212 space group with a 41 screw axis in a tetragonal arrangement, showing that the weakly bent DNA superhelices with a pitch length of ca. 5.0 nm possessed both identical handedness and phase conservation. The 3D ordered structure transformed into a 2D mesophase at a higher dendrimer charge density, wherein the strongly bent superhelices with a pitch length of ca. 4.0 nm organized in a hexagonal lattice without lateral coherence of helical trajectory. The counterion valency of the protonic acid that is used to charge the dendrimer was found to influence the phase diagram. Under a given dendrimer charge density, the complex with a multivalent acid-protonated dendrimer tended to form structures with less curved DNA, attesting that the driving force of charge matching was reduced by increasing the counterion valency of the dendrimer.

Corrigenda: SecA defects are accompanied by dysregulation of MukB, DNA gyrase, chromosome partitioning and DNA superhelicity in Escherichia coli.

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Requirements for DNA-Bridging Proteins to Act as Topological Barriers of the Bacterial Genome

Bacterial genomes have been shown to be partitioned into several-kilobase-long chromosomal domains that are topologically independent from each other, meaning that change of DNA superhelicity in one domain does not propagate to neighbors. Both in vivo and in vitro experiments have been performed to question the nature of the topological barriers at play, leading to several predictions on possible molecular actors. Here, we address the question of topological barriers using polymer models of supercoiled DNA chains that are constrained such as to mimic the action of predicted molecular actors. More specifically, we determine under which conditions DNA-bridging proteins may act as topological barriers. To this end, we developed a coarse-grained bead-and-spring model and investigated its properties through Brownian dynamics simulations. As a result, we find that DNA-bridging proteins must exert rather strong constraints on their binding sites; they must block the diffusion of the excess of twist through the two binding sites on the DNA molecule and, simultaneously, prevent the rotation of one DNA segment relative to the other one. Importantly, not all DNA-bridging proteins satisfy this second condition. For example, single bridges formed by proteins that bind DNA nonspecifically, like H-NS dimers, are expected to fail with this respect. Our findings might also explain, in the case of specific DNA-bridging proteins like LacI, why multiple bridges are required to create stable independent topological domains. Strikingly, when the relative rotation of the DNA segments is not prevented, relaxation results in complex intrication of the two domains. Moreover, although the value of the torsional stress in each domain may vary, their differential is preserved. Our work also predicts that nucleoid-associated proteins known to wrap DNA must form higher protein-DNA complexes to efficiently work as topological barriers.

A Dynamic Model of Superhelical DNA Denaturation

A Dynamic Model of Superhelical DNA Denaturation

Molecular dissection of Helicobacter pylori Topoisomerase I reveals an additional active site in the carboxyl terminus of the enzyme

DNA topoisomerases play a crucial role in maintaining DNA superhelicity, thereby regulating various cellular processes. Unlike most other species, the human pathogen Helicobacter pylori has only two topoisomerases, Topoisomerase I and DNA gyrase, the physiological roles of which remain to be explored. Interestingly, there is enormous variability among the C-terminal domains (CTDs) of Topoisomerase I across bacteria. H. pylori Topoisomerase I (HpTopoI) CTD harbors four zinc finger motifs (ZFs). We show here that sequential deletion of the third and/or fourth ZFs had only a marginal effect on the HpTopoI activity, while deletion of the second, third and fourth ZFs severely reduced DNA relaxation activity. Deletion of all ZFs drastically hampered DNA binding and thus abolished DNA relaxation. Surprisingly, mutagenesis of the annotated active site tyrosine residue (Y297 F) did not abrogate the enzyme activity and HpTopoI CTD alone (spanning the four ZFs) showed DNA relaxation activity. Additionally, a covalent linkage between the DNA and HpTopoI CTD was identified. The capacity of HpTopoI CTD to complement Escherichia coli topA mutant strains further supported the in vitro observations. Collectively these results imply that not all ZFs are dispensable for HpTopoI activity and unveil the presence of additional non-canonical catalytic site(s) within the enzyme.

Symmetry in Nucleic-Acid Double Helices

In nature and in the test tube, nucleic acids occur in many different forms. Apart from single-stranded, coiled molecules, DNA and RNA prefer to form helical arrangements, in which the bases are stacked to shield their hydrophobic surfaces and expose their polar edges. Focusing on double helices, we describe the crucial role played by symmetry in shaping DNA and RNA structure. The base pairs in nucleic-acid double helices display rotational pseudo-symmetry. In the Watson–Crick base pairs found in naturally occurring DNA and RNA duplexes, the symmetry axis lies in the base-pair plane, giving rise to two different helical grooves. In contrast, anti-Watson–Crick base pairs have a dyad axis perpendicular to the base-pair plane and identical grooves. In combination with the base-pair symmetry, the syn/anti conformation of paired nucleotides determines the parallel or antiparallel strand orientation of double helices. DNA and RNA duplexes in nature are exclusively antiparallel. Watson–Crick base-paired DNA or RNA helices display either right-handed or left-handed helical (pseudo-) symmetry. Genomic DNA is usually in the right-handed B-form, and RNA double helices adopt the right-handed A-conformation. Finally, there is a higher level of helical symmetry in superhelical DNA in which B-form double strands are intertwined in a right- or left-handed sense.

Cell Cycle-Dependent Control and Roles of DNA Topoisomerase II.

Type II topoisomerases are ubiquitous enzymes in all branches of life that can alter DNA superhelicity and unlink double-stranded DNA segments during processes such as replication and transcription. In cells, type II topoisomerases are particularly useful for their ability to disentangle newly-replicated sister chromosomes. Growing lines of evidence indicate that eukaryotic topoisomerase II (topo II) activity is monitored and regulated throughout the cell cycle. Here, we discuss the various roles of topo II throughout the cell cycle, as well as mechanisms that have been found to govern and/or respond to topo II function and dysfunction. Knowledge of how topo II activity is controlled during cell cycle progression is important for understanding how its misregulation can contribute to genetic instability and how modulatory pathways may be exploited to advance chemotherapeutic development.

Cyclometalated Gold(III) Complexes Bearing DACH Ligands.

The synthesis of a novel class of cyclometalated gold(III) complexes supported by benzoylpyridine, benzylpyridine, and (1R,2R)-(+)-1,2-diaminocyclohexane (DACH) ligands, along with their crystal structures, is reported. These compounds provide a new scaffold to investigate biological properties of gold(III) complexes. The six complexes were prepared and characterized, following reactions of (C,N) cyclometalated gold(III) scaffolds, [Au(C^N)Cl2] with DACH, which yielded a new series of cyclometaled gold(III), 3-5, of the type [Au(C^NH)(DACH)2]+ and the nitrogen-substituted cyclometalated Au(III), 6-8, of the type [Au(C^N)(DACH)]2+. Antiproliferative activity of these complexes in a panel of cancer cells showed promising results with IC50 in the micromolar range and selectivity over normal epithelial cells, MRC5. Whereas 8 shows minimal interaction with superhelical DNA except at high gold concentrations of 500 μM, complex 5 does not show interaction even at 1000 μM. The complexes display significant uptake in OVCAR8 cancer cells within 200-1200 pmol/million cells with the exception of complex 4. Differential cellular uptake was observed for the complexes; for example, while 3 and 8 display significant uptake, 4 showed minimal uptake. The compounds proved to be stable under physiological conditions and were minimally affected by either glutathione or sodium ascorbate. Cell cycle studies reveal a G1 arrest induced by representative complexes. The results reveal that enhanced Au(III) stabilization promoted by combined cyclometalated and DACH ligands may offer ligand tuning insights for novel anticancer drug design.

Interplay between DNA sequence and negative superhelicity drives R-loop structures

R-loops are abundant three-stranded nucleic-acid structures that form in cis during transcription. Experimental evidence suggests that R-loop formation is affected by DNA sequence and topology. However, the exact manner by which these factors interact to determine R-loop susceptibility is unclear. To investigate this, we developed a statistical mechanical equilibrium model of R-loop formation in superhelical DNA. In this model, the energy involved in forming an R-loop includes four terms-junctional and base-pairing energies and energies associated with superhelicity and with the torsional winding of the displaced DNA single strand around the RNA:DNA hybrid. This model shows that the significant energy barrier imposed by the formation of junctions can be overcome in two ways. First, base-pairing energy can favor RNA:DNA over DNA:DNA duplexes in favorable sequences. Second, R-loops, by absorbing negative superhelicity, partially or fully relax the rest of the DNA domain, thereby returning it to a lower energy state. In vitro transcription assays confirmed that R-loops cause plasmid relaxation and that negative superhelicity is required for R-loops to form, even in a favorable region. Single-molecule R-loop footprinting following in vitro transcription showed a strong agreement between theoretical predictions and experimental mapping of stable R-loop positions and further revealed the impact of DNA topology on the R-loop distribution landscape. Our results clarify the interplay between base sequence and DNA superhelicity in controlling R-loop stability. They also reveal R-loops as powerful and reversible topology sinks that cells may use to nonenzymatically relieve superhelical stress during transcription.

Architectural Organization of Dinoflagellate Liquid Crystalline Chromosomes.

Dinoflagellates have some of the largest genome sizes, but lack architectural nucleosomes. Their liquid crystalline chromosomes (LCCs) are the only non-architectural protein-mediated chromosome packaging systems, having high degrees of DNA superhelicity, liquid crystalline condensation and high levels of chromosomal divalent cations. Recent observations on the reversible decompaction–recompaction of higher-order structures implicated that LCCs are composed of superhelical modules (SPMs) comprising highly supercoiled DNA. Orientated polarizing light photomicrography suggested the presence of three compartments with different packaging DNA density in LCCs. Recent and previous biophysical data suggest that LCCs are composed of: (a) the highly birefringent inner core compartment (i) with a high-density columnar-hexagonal mesophase (CH-m); (b) the lower-density core surface compartment (ii.1) consisting of a spiraling chromonema; (c) the birefringent-negative periphery compartment (ii.2) comprising peripheral chromosomal loops. C(ii.1) and C(ii.2) are in dynamic equilibrium, and can merge into a single compartment during dinomitosis, regulated through multiphasic reversible soft-matter phase transitions.

The Hanahan Method for Preparation and Transformation of Competent Escherichia coli: High-Efficiency Transformation.

Chemical transformation is a highly efficient, commonly used method to transform Escherichia coli with plasmid DNA. Hanahan's procedure works well with many strains of E. coli commonly used in molecular cloning. Many variations on the basic technique have been described, all directed toward optimizing the efficiency of transformation. It is now possible on a routine basis to achieve transformation frequencies ranging from 106 to 109 transformants/µg of superhelical plasmid DNA.

Nucleosome stability measured in situ by automated quantitative imaging

Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

Synthesis, chemical nuclease activity, and in vitro cytotoxicity of benzimidazole-based Cu(II)/Co(II) complexes

In this study, novel mono/di-nuclear Cu(p-2-bmb)(OH)(ClO4) (1) and Co2(p‐2-bmb)2Cl4 (2) (p-2-bmb=1-((2-(pyridin-2-yl)-benzoimidazol-1-yl)methyl)-1H-benzotriazole) complexes with the nitrogen heterocyclic benzimidazole-based ligand were synthesized and characterized. The two complexes showed anti-proliferative effects in various carcinoma cell lines, especially complex 1 in the SMMC7721 tumor cell line. Complex 1 was also able to pass through the cell membrane and enter the nucleus and mitochondrion. An analysis of in vitro chemical nuclease activity revealed that complex 1 partially intercalated to calf thymus DNA and exhibited strong unwinding activity against pBR322 superhelical plasmid DNA. The comet assay and flow cytometry analysis confirmed that 1 caused extensive DNA damage and arrested SMMC7721 tumor cells at G2/M phase of the cell cycle, leading to loss of mitochondrial membrane potential and apoptosis. These results suggest that these benzimidazole-based metal complexes could be potential anti-cancer agents.

S1-Type Endonuclease 2 in Dedifferentiating Arabidopsis Protoplasts: Translocation to the Nucleus in Senescing Protoplasts Is Associated with De-Glycosylation.

Cell dedifferentiation characterizes the transition of leaf cells to protoplasts and is accompanied by global chromatin decondensation. Here we show that in Arabidopsis, chromocentric chromatin undergoes prompt and gradual decondensation upon protoplasting. We hypothesized that prompt chromatin decondensation is unlikely to be driven solely by epigenetic means and other factors might be involved. We investigated the possibility that S1-type endonucleases are involved in prompt chromatin decondensation via their capability to target and cleave unpaired regions within superhelical DNA, leading to chromatin relaxation. We showed that the expression and activity of the S1-type endonuclease 2 (ENDO2) is upregulated in dedifferentiating protoplasts concomitantly with chromatin decondensation. Mutation of the ENDO2 gene did not block or delay chromocentric chromatin decondensation upon protoplasting. Further study showed that ENDO2 subcellular localization is essentially cytoplasmic (endoplasmic reticulum-associated) in healthy cells, but often localized to the nucleus and in senescing/dying cells it was associated with fragmented nuclei. Using in gel nuclease assays we identified two ENDO2 variants, designated N1 (cytoplasmic variant) and N2 (cytoplasmic and nuclear variant), and based on their capability to bind concanavalin A (ConA), they appear to be glycosylated and de-glycosylated (or decorated with ConA non-binding sugars), respectively. Our data showed that the genome is responding promptly to acute stress (protoplasting) by acquiring decondensation state, which is not dependent on ENDO2 activity. ENDO2 undergoes de-glycosylation and translocation to the nucleus where it is involved in early stages of cell death probably by introducing double strand DNA breaks into superhelical DNA leading to local chromatin relaxation and fragmentation of nuclei.