DNA Stiffness Research Articles

A luminescent europium hairpin for DNA photosensing in the visible, based on trimetallic bis-intercalators

Luminescence monitoring of DNA intercalator complexes is important for assessing their localisation and targeting: We report herein a luminescent hetero-trimetallic complex with europium as a luminescent reporter and two attached platinum acetylide terpyridyl units as the DNA recognition units. The ligand, based on a bisamide derivative of diethylenetriaminepentaacetic acid functionalized with two ethynyl groups, provides a backbone to anchor two platinum terpyridyl units, Pt-tpy, leading to the hairpin-shaped heterometallic complex 1. We also prepared a related mono-nuclear platinum complex 2 to compare its intercalation properties with 1. Linear dichroism, UV–visible and melting experiments show the ability of both complexes to interact with calf thymus DNA, with linear dichroism confirming intercalation and demonstrating the expected greater DNA stiffening by the bis-intercalator 1. Importantly, the tri-metallic complex 1 shows a three-fold enhancement in europium luminescence upon addition of calf thymus DNA; other mono-intercalator lanthanide designs have commonly shown a decrease in emission on binding. The ability of the complex to monitor DNA interactions gives the potential use as a luminescence switch in sensing experiments and highlights the design of heterometallic bis-intercalator complexes as an effective approach for DNA-responsive sensitisation of a lanthanide luminescence signal.

Effect of Methylation on Local Mechanics and Hydration Structure of DNA

Cytosine methylation affects mechanical properties of DNA and potentially alters the hydration fingerprint for recognition by proteins. The atomistic origin for these effects is not well understood, and we address this via all-atom molecular dynamics simulations. We find that the stiffness of the methylated dinucleotide step changes marginally, whereas the neighboring steps become stiffer. Stiffening is further enhanced for consecutively methylated steps, providing a mechanistic origin for the effect of hypermethylation. Steric interactions between the added methyl groups and the nonpolar groups of the neighboring nucleotides are responsible for the stiffening in most cases. By constructing hydration maps, we found that methylation also alters the surface hydration structure in distinct ways. Its resistance to deformation may contribute to the stiffening of DNA for deformational modes lacking steric interactions. These results highlight the sequence- and deformational-mode-dependent effects of cytosine methylation.

Elucidating the mechanism of the considerable mechanical stiffening of DNA induced by the couple Zn2+/Calix[4]arene-1,3-O-diphosphorous acid

The couple Calix[4]arene-1,3-O-diphosphorous acid (C4diP) and zinc ions (Zn2+) acts as a synergistic DNA binder. Silicon NanoTweezer (SNT) measurements show an increase in the mechanical stiffness of DNA bundles by a factor of >150, at Zn2+ to C4diP ratios above 8, as compared to Zinc alone whereas C4diP alone decreases the stiffness of DNA. Electroanalytical measurements using 3D printed devices demonstrate a progression of events in the assembly of C4diP on DNA promoted by zinc ions. A mechanism at the molecular level can be deduced in which C4diP initially coordinates to DNA by phosphate-phosphate hydrogen bonds or in the presence of Zn2+ by Zn2+ bridging coordination of the phosphate groups. Then, at high ratios of Zn2+ to C4diP, interdigitated dimerization of C4diP is followed by cross coordination of DNA strands through Zn2+/C4diP inter-strand interaction. The sum of these interactions leads to strong stiffening of the DNA bundles and increased inter-strand binding.

Force and Scale Dependence of the Elasticity of Self-Assembled DNA Bottle Brushes.

As a model system to study the elasticity of bottle-brush polymers, we here introduce self-assembled DNA bottle brushes, consisting of a DNA main chain that can be very long and still of precisely defined length, and precisely monodisperse polypeptide side chains that are physically bound to the DNA main chains. Polypeptide side chains have a diblock architecture, where one block is a small archaeal nucleoid protein Sso7d that strongly binds to DNA. The other block is a net neutral, hydrophilic random coil polypeptide with a length of exactly 798 amino acids. Light scattering shows that for saturated brushes the grafting density is one side chain per 5.6 nm of DNA main chain. According to small-angle X-ray scattering, the brush diameter is D = 17 nm. By analyzing configurations of adsorbed DNA bottle brushes using AFM, we find that the effective persistence of the saturated DNA bottle brushes is Peff = 95 nm, but from force–extension curves of single DNA bottle brushes measured using optical tweezers we find Peff = 15 nm. The latter is equal to the value expected for DNA coated by the Sso7d binding block alone. The apparent discrepancy between the two measurements is rationalized in terms of the scale dependence of the bottle-brush elasticity using theory previously developed to analyze the scale-dependent electrostatic stiffening of DNA at low ionic strengths.

A stochastic reaction-diffusion model for protein aggregation on DNA

Vital functions of DNA, such as transcription and packaging, depend on the proper clustering of proteins on the double strand. The present study investigates how the interplay between DNA allostery and electrostatic interactions affects protein clustering. The statistical analysis of a simple but transparent computational model reveals two major consequences of this interplay. First, depending on the protein and salt concentration, protein filaments exhibit a bimodal DNA stiffening and softening behavior. Second, within a certain domain of the control parameters, electrostatic interactions can cause energetic frustration that forces proteins to assemble in rigid spiral configurations. Such spiral filaments might trigger both positive and negative supercoiling, which can ultimately promote gene compaction and regulate the promoter. It has been experimentally shown that bacterial histone-like proteins assemble in similar spiral patterns and/or exhibit the same bimodal behavior. The proposed model can, thus, provide computational insights into the physical mechanisms used by proteins to control the mechanical properties of the DNA.

Atomic force microscopy of chromatin arrays reveal non-monotonic salt dependence of array compaction in solution.

Compaction of DNA in chromatin is a hallmark of the eukaryotic cell and unravelling its structure is required for an understanding of DNA involving processes. Despite strong experimental efforts, many questions concerning the DNA packing are open. In particular, it is heavily debated whether an ordered structure referred to as the “30 nm fibre” exist in vivo. Scanning probe microscopy has become a cutting edge technology for the high-resolution imaging of DNA- protein complexes. Here, we perform high-resolution atomic force microscopy of non-cross-linked chromatin arrays in liquid, under different salt conditions. A statistical analysis of the data reveals that array compaction is salt dependent in a non-monotonic fashion. A simple physical model can qualitatively explain the observed findings due to the opposing effects of salt dependent stiffening of DNA, nucleosome stability and histone-histone interactions. While for different salt concentrations different compaction states are observed, our data do not provide support for the existence of regular chromatin fibres. Our studies add new insights into chromatin structure, and with that contribute to a further understanding of the DNA condensation.

Locus of Enterocyte Effacement-encoded Regulator (Ler) of Pathogenic Escherichia coli Competes Off Histone-like Nucleoid-structuring Protein (H-NS) through Noncooperative DNA Binding

The locus of enterocyte effacement-encoded regulator (Ler) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) functions to activate transcription of virulence genes silenced by the histone-like nucleoid-structuring protein (H-NS). Despite its important role in the bacterial gene regulation, the binding mode of Ler to DNA and its mechanism in alleviating genes repressed by H-NS are largely unknown. In this study, we use magnetic tweezers to demonstrate that Ler binds extended DNA through a largely noncooperative process, which results in DNA stiffening and DNA folding depending on protein concentration. We also show that Ler can replace prebound H-NS on DNA over a range of potassium and magnesium concentrations. Our findings reveal the DNA binding properties of Ler and shed light to further understand the anti-silencing activity of Ler.

Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.

Relation between the change in DNA elasticity on ligand binding and the binding energetics

The widespread use of tweezers for measurement of ligand-DNA binding parameters is based on the McGhee-von Hippel treatment of the DNA contour and persistence length as a function of concentration. The McGhee-von Hippel approach contains the basic assumption that the binding constant K is independent of the number of already bound ligands. However, the change in elasticity of DNA on binding affects the entropic part of the Gibbs free energy and, hence, the K value in a concentration-dependent manner, making the whole approach inconsistent. In the present work we show that the energetic effect of DNA stiffening on noncovalent binding of small ligands is negligible with respect to the net energy of reaction, whereas the DNA stiffening on binding of large ligands must always be considered in each particular case.

Effective stiffening of DNA due to nematic ordering causes DNA molecules packed in phage capsids to preferentially form torus knots

Observation that DNA molecules in bacteriophage capsids preferentially form torus type of knots provided a sensitive gauge to evaluate various models of DNA arrangement in phage heads. Only models resulting in a preponderance of torus knots could be considered as close to reality. Recent studies revealed that experimentally observed enrichment of torus knots can be qualitatively reproduced in numerical simulations that include a potential inducing nematic arrangement of tightly packed DNA molecules within phage capsids. Here, we investigate what aspects of the nematic arrangement are crucial for inducing formation of torus knots. Our results indicate that the effective stiffening of DNA by the nematic arrangement not only promotes knotting in general but is also the decisive factor in promoting formation of DNA torus knots in phage capsids.

Is DNA’s Rigidity Dominated by Electrostatic or Nonelectrostatic Interactions?

Double-stranded DNA is among the stiffest biopolymers, whose bending propensity crucially influences many vital biological processes. It is not fully understood which among the two most likely forces, electrostatic self-repulsion or the compressive base pair stacking, plays a dominant role in determining the DNA's unique rigidity. Different theoretical and experimental studies led so far to contradictory results on this issue. In this Communication, we address this important question by means of Molecular Dynamics (MD) simulations using both atomistic and coarse-grained force fields. Using two independent sets of calculations, we found that electrostatic and nonelectrostatic effects play a comparable role in maintaining DNA's stiffness. Our findings substantially differ from predictions of existing theories for DNA rigidity and may indicate that a new conceptual understanding needs to be developed.

Nucleic‐acid structural deformability deduced from anisotropic displacement parameters

The growing numbers of very well resolved nucleic-acid crystal structures with anisotropic displacement parameters provide an unprecedented opportunity to learn about the natural motions of DNA and RNA. Here we report a new Monte-Carlo approach that takes direct account of this information to extract the distortions of covalent structure, base pairing, and dinucleotide geometry intrinsic to regularly organized double-helical molecules. We present new methods to test the validity of the anisotropic parameters and examine the apparent deformability of a variety of structures, including several A, B, and Z DNA duplexes, an AB helical intermediate, an RNA, a ligand-DNA complex, and an enzyme-bound DNA. The rigid-body parameters characterizing the positions of the bases in the structures mirror the mean parameters found when atomic motion is taken into account. The base-pair fluctuations intrinsic to a single structure, however, differ from those extracted from collections of nucleic-acid structures, although selected base-pair steps undergo conformational excursions along routes suggested by the ensembles. The computations reveal surprising new molecular insights, such as the stiffening of DNA and concomitant separation of motions of contacted nucleotides on opposite strands by the binding of Escherichia coli endonuclease VIII, which suggest how the protein may direct enzymatic action.

A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes

Heat-stable nucleoid structuring protein (H-NS) is an abundant prokaryotic protein that plays important roles in organizing chromosomal DNA and gene silencing. Two controversial binding modes were identified. H-NS binding stimulating DNA bridging has become the accepted mechanism, whereas H-NS binding causing DNA stiffening has been largely ignored. Here, we report that both modes exist, and that changes in divalent cations drive a switch between them. The stiffening form is present under physiological conditions, and directly responds to pH and temperature in vitro. Our findings have broad implications and require a reinterpretation of the mechanism by which H-NS regulates genes.

Illuminating the DNA Binding Behavior of Mitochondrial Transcription Factor A

Mitochondria are the energy producing organelles of eukaryotic cells. Owing to their endosymbiotic evolutionary history, they contain their own genome (mtDNA) that encodes for thirteen proteins essential for ATP production.In mammalian cells, multiple mtDNAs are compacted into protein-DNA complexes called “nucleoids”. A major component of these nucleoids is the mitochondrial transcription factor A (TFAM), a member of the high mobility group (HMG) family of proteins. This abundant protein binds DNA with little sequence specificity, and is able to coat the entire mtDNA molecule. It not only serves a role in mtDNA packaging, but is also required for mitochondrial transcription. At this point, dynamics of the TFAM-DNA interaction remain unclear.Experiments on single DNA molecules offer a very direct way to study TFAM dynamics. Tethered Particle Motion (TPM) experiments show that the system quickly equilibrates with the buffer, and that the end-to-end distance of the DNA decreases upon TFAM binding. Manipulations of single DNA molecules with two optical traps offer an explanation: TFAM decreases the DNA's stiffness (persistence length). A possible molecular mechanism for this decrease is that TFAM introduces bends in the DNA.Adding single-molecule fluorescence to the dual optical trap illuminates TFAM's binding behavior. Literally seeing TFAM on the DNA, we can derive on- and off-rates, and determine that TFAM does not bind cooperatively. Interestingly and seemingly unrelated to its role in DNA organization, we also observe that TFAM can rapidly bind single-stranded DNA (ssDNA), but not when the ssDNA is under tension. The physiological function of TFAM binding to ssDNA could be related to its regulation of transcription.

DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum

Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasite's intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of ∼27 bp and a Kd of ∼63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU–DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.

Ss-LrpB from Sulfolobus solfataricus Condenses about 100 Base Pairs of Its Own Operator DNA into Globular Nucleoprotein Complexes

Ss-LrpB from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2 is a member of the Lrp-like family of Bacterial/Archaeal transcription regulators that binds its own control region at three regularly spaced and partially conserved 15-bp-long imperfect palindromes. We have used atomic force microscopy to analyze the architecture of Ss-LrpB.DNA complexes with a different stoichiometry formed with the wild type operator and with an operator mutant. Binding of dimeric Ss-LrpB to all three target sites is accompanied by the formation of globular complexes, in which the protein induces strong DNA deformations. Furthermore, DNA contour length foreshortening of these complexes indicates DNA wrapping, with about 100 bp being condensed. The average bending angle is 260 degrees . The establishment of protein-protein contacts between Ss-LrpB dimers in these globular complexes will contribute to the cooperativity of the binding. The profound remodeling of the control region is expected to have a strong impact on gene expression and might constitute the key element in the autoregulatory process.

Hairpin-shaped heterometallic luminescent lanthanide complexes for DNA intercalative recognition.

Luminescent Ln-Pt2 metallohairpin complexes have been developed, and their intercalative recognition with DNA has been demonstrated with linear dichroism spectroscopy. The heterotrimetallic complexes were formed in a one-step reaction, by assembly of an aminopolycarboxylate ligand, a platinum terpyridine unit, and the lanthanide salt. The metallohairpin complexes bear a neutral lanthanide moiety and two positively charged platinum-containing intercalating units. The Nd(III) analogues are luminescent in the near infrared, and this near-IR luminescence is retained upon binding to DNA. The DNA recognition was demonstrated by linear dichroism spectroscopy. The linear dichroism spectra suggested that the complexes bind perpendicular to the DNA helical axis, confirming intercalative recognition accompanied by dramatic stiffening of DNA, which suggests bis-intercalation of the complex.

DNA-Helix Bending, Stiffening and Elongation on Ligand Binding; Analysis for Serveral DNA-Drug Systems, General Viscometric DNA Response and Stereochemical Implications

For several DNA-ligand systems the DNA helix bending, stiffening and elongation behaviour is treated quantitatively. The experimental basis are viscosity data from literature as a function of r, the ratio of drug molecules bound per DNA monomer unit. If the relative viscosity changes delta y1(r) and delta yh(r) for DNA of low and high molar mass, respectively, are known, the relative changes of contour length, delta L/L degrees, and of persistence length, delta a/a degrees, can be evaluated as a function of r, as repeatedly demonstrated. For random sequence-independent interactions, helix-bending is reflected by a helix-typical increment of delta a/a degrees (r), being zero at r = 0 and also at DNA saturation by bound ligand molecules [Reinert, Biophysical Chemistry 13, 1-14 (1981)]. This characteristic DNA behaviour often enables us to separate the bending and the stiffening increment of delta a/a degrees. The theoretical treatment of this problem (Schütz and Reinert, J. Biomolec. Struct. & Dynam. 9, 315-329, 1991) now permits a more detailed study of the ligand-induced DNA bending. The ligand-DNA systems treated here concern the following drugs (in parentheses DNA bending angle at low r-values): proflavin (8 degrees), daunomycin (11 degrees), aclacinomycin A (9.7 degrees, on cooperative interaction), actinomycin D (16 degrees), mitomycin C (16 degrees), a double intercalating bisphenantridine (12 degrees), 9-deacetyl-daunomycin (8 degrees) and 9-epi-deacetyl-daunomycin (12-18 degrees). We also demonstrate that the consideration of the DNA flexibility and its change on interaction of short DNA molecules with intercalating drugs delivers helix elongation values in better accord with the theoretical value. In the Appendix, a catalogue of simulated delta y(r)-dependences is given for both short and long DNA molecules. It systematically describes the DNA viscosity response upon typical DNA stiffening, elongation, and helix-bending effects.

Determination of the structural features of (+)-CC-1065 that are responsible for bending and winding of DNA

Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity, [Hurley, L. H., Lee, C.-S., McGovern, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19 degrees, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5'-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered.

Influence of DNA length on spermine-induced condensation: Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.