Opening Of Helix Research Articles

Topological solitons in an inhomogeneous DNA molecule

The dynamics of topological solitons describing the opening of the double helix of a DNA molecule is studied. The estimated actual values of the rigidity of the polynucleotide chains made it possible to develop a more precise DNA model and to show that four types of topological solitons can appear in the DNA double helix. Interactions between solitons are studied, as well as their interaction with the chain inhomogeneities and the stability of solitons with respect to thermal fluctuations. Thermal fluctuations promote propagation of solitons along an inhomogeneous base sequence.

Conformational change of apolipophorin III upon lipopolysaccharide binding

Apolipophorin III (apoLp-III) from the Greater wax moth, Galleria mellonella, is an exchangeable apolipoprotein. ApoLp-III functions in the stabilization of low-density lipophorin particles which are used to mobilize lipids. The protein is also able to bind to lipopolysaccharides (LPS), and may function as a pattern recognition protein. ApoLp-III is organized as a five α-helical bundle with a simple up-and-down topology. Lipid binding is accompanied by an opening of the helix bundle. We hypothesize that a similar conformational change in apoLp-III occurs upon LPS binding. To prevent helix bundle opening, four cysteine residues were introduced to allow formation of two disulfide bridges to tether the helices. Cysteine pairs were engineered at position 5 on Helix (H) 1 and at position 135 on H5, and at position 37 on H2 and position 95 on H3 (P5C/A135C/N37C/E95C apoLp-III). Mutant proteins were expressed in a bacterial _expression system, and purified by HPLC. Binding studies, using intrinsic tyrosine fluorescence and gel shift assays will be used to address the helix opening hypothesis. Proteins will be studied under oxidizing and reducing conditions to control helix bundle opening. Two other mutant apolipoproteins that have been shown to display decreased lipid binding will also be tested. These proteins, with mutations in the second loop, will be used to investigate the similarity between lipid and LPS binding.

Interaction of Antioxidant Flavonoids with tRNA: Intercalation or External Binding and Comparison with Flavonoid-DNA Adducts

Antioxidants are essential to good health. Flavonoids are powerful antioxidants, and prevent DNA damage. The antioxidative protections are related to their binding modes to a DNA duplex and complexation with free radicals in vivo. Recently we reported the interaction of flavonoids with DNA in vitro (Kanakis et al., J. Biomol. Struct. Dyn. 22, 719-724, 2005), where polyphenol different binding modes were discussed. The aim of this study was to examine the interaction of transfer RNA with quercetin (que), kaempferol (kae), and delphinidin (del) in aqueous solution at physiological conditions and to make a comparison with the corresponding pigment-DNA adducts. Constant tRNA concentration (6.25 mM) and various drug/RNA(phosphate) molar ratios of 1/48 to 1/8 were used. FTIR and UV-visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants, and the effects of drug complexation on the stability and conformation of tRNA duplex. Both intercalative and external binding modes were observed. Structural analysis showed que, kae, and a del intercalate tRNA duplex with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants K (que) = 4.80 x 10(4) M(1), K (kae) = 4.65 x 10(4) M(1), and K (del) = 9.47 x 10(4) M(1). The stability of adduct formation is in the order of del > que > kae. A comparison with flavonoids-DNA adducts showed both intercalation and external bindings with the stability order K (que) = 7.25 x 10(4) M(1), K (kae) = 3.60 x 10(4) M(1), and K (del) = 1.66 x 10(4) M(1). Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial Bto A-DNA transition occurs at high drug concentration, while tRNA remains in the A-family structure.

Molecular Dynamics Simulations Reveal Multiple Pathways of Ligand Dissociation from Thyroid Hormone Receptors

Nuclear receptor (NR) ligands occupy a pocket that lies within the core of the NR ligand-binding domain (LBD), and most NR LBDs lack obvious entry/exit routes upon the protein surface. Thus, significant NR conformational rearrangements must accompany ligand binding and release. The precise nature of these processes, however, remains poorly understood. Here, we utilize locally enhanced sampling (LES) molecular dynamics computer simulations to predict molecular motions of x-ray structures of thyroid hormone receptor (TR) LBDs and determine events that permit ligand escape. We find that the natural ligand 3,5,3′-triiodo-L-thyronine (T 3) dissociates from the TR α1 LBD along three competing pathways generated through i), opening of helix (H) 12; ii), separation of H8 and H11 and the Ω-loop between H2 and H3; and iii), opening of H2 and H3, and the intervening β-strand. Similar pathways are involved in dissociation of T 3 and the TR β-selective ligand GC24 from TR β; the TR agonist IH5 from the α- and β-TR forms; and Triac from two natural human TR β mutants, A317T and A234T, but are detected with different frequencies in simulations performed with the different structures. Path I was previously suggested to represent a major pathway for NR ligand dissociation. We propose here that Paths II and III are also likely ligand escape routes for TRs and other NRs. We also propose that different escape paths are preferred in different situations, implying that it will be possible to design NR ligands that only associate stably with their cognate receptors in specific cellular contexts.

Covalent binding of antitumor benzoacronycines to double-stranded DNA induces helix opening and the formation of single-stranded DNA: Unique consequences of a novel DNA-bonding mechanism

The majority of DNA-binding small molecules known thus far stabilize duplex DNA against heat denaturation. A high, drug-induced increase in the melting temperature (Tm) of DNA is generally viewed as a good criterion to select DNA ligands and is a common feature of several anticancer drugs such as intercalators (e.g., anthracyclines) and alkylators (e.g., ecteinascidin 743). The reverse situation (destabilization of DNA to facilitate its denaturation) may be an attractive option for the identification of therapeutic agents acting on the DNA structure. We have identified the tumor-active benzoacronycine derivative S23906-1 [(+/-)-cis-1,2-diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2]acridin-7-one] as a potent DNA alkylating agent endowed with a helicase-like activity. Using complementary molecular approaches, we show that covalent binding to DNA of the diacetate compound S23906-1 and its monoacetate analogue S28687-1 induces a marked destabilization of the double helix with the formation of alkylated ssDNA. The DNA-bonding properties and effects on DNA structure of a series of benzoacronycine derivatives, including the dicarbamate analogue S29385-1, were studied using complementary biochemical (electromobility shift assay, nuclease S1 mapping) and spectroscopic (fluorescence and Tm measurements) approaches. Alkylation of guanines in DNA by S28687-1 leads to a local denaturation of DNA, which becomes susceptible to cleavage by nuclease S1 and significantly decreases the Tm of DNA. The drug also directly alkylates single-strand DNA, but mass spectrometry experiments indicate that guanines in duplexes are largely preferred over single-stranded structures. This molecular study expands the repertoire of DNA-binding mechanisms and provides a new dimension for DNA recognition by small molecules.

Impact of Radiation Damage on DNA Determined by Computational Simulation

Review of molecular dynamics (MD) studies of several radiation-originated lesions on DNA molecules is presented. Main focus is to describe structural and energy changes in DNA molecule with the respect to proper recognition of the lesion by respective repair enzyme. Pyrimidine lesions (cytosinyl radical, thymine glycol) and purine lesion (8-oxoguanine) were subjected to MD simulations for several hundreds picosecond (ps), (200ps for cytosinyl radical, 2 nanosecond for thymine glycol and 8-oxoguanine) using MD simulation code AMBER 5.0 (4.0) and its respective force field modified for each lesion. Simulations were performed as all atoms simulations for fully solvated solute molecules in water. Negative charges of DNA phosphates were neutralized by sodium counterions Na+ that are essential for its double helical structure. In most cases the significant structural changes in DNA are observed: a) breaking of hydrogen bonds network between complementary bases and resulting opening of the double helix (cytosinyl radical, 8-oxoguanine); b) sharp bending of the DNA helix centered at the lesion site (thymine glycol); and c) flipped-out base (8-oxoguanine). These changes are related to overall collapsing of the double helical structure around the lesion and are considered to facilitate docking of the repair enzyme into the DNA and formation of DNA-enzyme complex. Stable DNA-enzyme complex is necessary condition for the onset of entire enzymatic repair process. In addition to the structural changes, specific values of electrostatic interaction energy are detected at several lesion sites (thymine glycol, 8-oxoguanine). The specific electrostatic energy is considered as a factor that enables repair enzyme to discriminate lesion from native, non-damaged site.

Structural changes in lumirhodopsin and metarhodopsin I studied by their photoreactions at 77 K.

The functional process of rhodopsin is initiated by cis-trans photoisomerization of the retinal chromophore. One of the primary intermediates, bathorhodopsin (Batho), is stable at 77 K, and structural changes in Batho are limited around the chromophore. Then, relaxation of Batho leads to helix opening at the cytoplasmic surface in metarhodopsin II (Meta II), which allows activation of a G protein transducin. Two intermediates, lumirhodopsin (Lumi) and metarhodopsin I (Meta I), appear between Batho and Meta II, and can be stabilized at 200 and 240 K, respectively. A photoaffinity labeling experiment reported that formation of Lumi accompanied flip-over of the beta-ionone ring of the retinal chromophore so that the ring portion was attached to Ala169 of helix IV [Borhan, B., Souto, M. L., Imai, H., Shichida, Y., and Nakanishi, K. (2000) Science 288, 2209-2212]. According to the crystal structure of bovine rhodopsin, the distance between the labeled C3 atom of the chromophore and Ala169 was >15 A [Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745]. These facts suggest that global protein structural changes such as helix motions take place in Lumi. In the study presented here, Lumi and Meta I are illuminated at 77 K, and protein structural changes are probed by Fourier transform infrared (FTIR) spectroscopy. We found that Lumi can be photoconverted to rhodopsin at 77 K from the IR spectral analysis of the photoproducts of Lumi. In contrast, more complex spectra were obtained for the photoproducts of Meta I at 77 K, implying that the protein structure of Meta I is considerably altered so as not to be reverted to the original state at 77 K. Thus, these photoreaction experiments with Lumi and Meta I at 77 K suggested the presence of global protein structural changes in the process between them. We concluded that the helix motions do not occur at Lumi, but at Meta I, and the flip-over of the beta-ionone ring reported by the photoaffinity labeling takes place through the specific reaction channel without a change in the global structure.

Computational determination of radiation damage effects on DNA structure

AbstractMolecular dynamics (MD) studies of several radiation originated lesions on the DNA molecules are presented. The pyrimidine lesions (cytosinyl radical, thymine dimer, thymine glycol) and purine lesion (8-oxoguanine) were subjected to the MD simulations for several hundred picoseconds using MD simulation code AMBER 5.0 (4.0). The simulations were performed for fully dissolved solute molecules in water. Significant structural changes in the DNA double helical structure were observed in all cases which may be categorized as: a) the breaking of hydrogen bonds network between complementary bases and resulted opening of the double helix (cytosinyl, radical, 8-oxoguanine); b) the sharp bending of the DNA helix centered at the lesion site (thymine dimer, thymine glycol); and c) the flippingout of adenine on the strand complementary to the lesion (8-oxoguanine). These changes related to the overall collapsing of the double helical structure around the lesion, are expected to facilitate the docking of the repair enzyme into the DNA in the formation of DNA-enzyme complex. The stable DNA-enzyme complex is a necessary condition for the onset of the enzymatic repair process. In addition to structural changes, specific values of electrostatic interaction energy were determined at several lesion sites (thymine dimer, thymine glycol and 8-oxoguanine). This lesion-specific electrostatic energy is a factor that enables repair enzyme to discriminate lesion from the native site during the scanning of the DNA surface.

Rotational drag on DNA: a single molecule experiment.

Within a single-molecule configuration, we have studied rotational drag on double stranded linear DNA by measuring the force during mechanical opening and closing of the double helix at different rates. The molecule is cranked at one end by the effect of unzipping and is free to rotate at the other end. In this configuration the rotational friction torque tau on double-stranded DNA leads to an additional contribution to the opening force. It is shown that the effect of rotational drag increases with the length of the molecule, is approximately proportional to the angular velocity of cranking, and we estimate that the torque tau is of the order of 1k(B)T for 10 000 base pairs of DNA cranked at 2000 turns per second.

Mechanical opening of DNA by micro manipulation and force measurements

In this paper we summarize part of our work on the mechanical unzipping of DNA. We have prepared molecular constructions which allow us to attach the two complementary strands of one end of a single DNA molecule of the bacteriophage λ separately to a glass microscope slide and a microscopic bead. In a first series of experiments, a soft microneedle acting as a force sensor is attached to the bead and its deflection is measured with an optical microscope. In a second series, we use an optical trapping interferometer to capture the bead and to measure its displacement to nm resolution. The sample is slowly displaced with respect to the force measurement device, leading to a progressive opening of the double helix. The force measured during this mechanical opening shows a characteristic variation which is related to the base pair sequence of the DNA molecule. To cite this article: U. Bockelmann et al., C. R. Physique 3 (2002) 585–594.

Thermodynamics of local DNA openings.

A mechanism connecting the local untwisting and opening of DNA double helix is proposed. The presented thermodynamical approach is based on two models: the Peyrard-Bishop model that describes the denaturation of DNA due to thermal fluctuations and the model developed by the author describing solitary torsional waves, which propagate along the DNA molecule forced by advancing RNA polymerase. The torsional wave implies that the DNA untwists locally causing a local decrease in the stacking interaction between adjacent base pairs. Molecular dynamics simulations have shown that thermal fluctuations (which are too small at physiological temperatures to denaturate the twisted DNA) may lead to the formation of a denaturation bubble placed in the untwisted region.

Calf-Thymus DNA Interaction with Cr(III)-Gallate and Cr(III)-Ethyl Gallate Studied by FTIR Spectroscopy and Capillary Electrophoresis

Abstract Cr(VI) salts are well known to be carcinogen, and are reduced by various cellular components to form cross-linked Cr(III) products. Organic compounds, such as gallic acid (GA) and ethyl gallate (EGA), reduce Cr(VI) to Cr(III) to form Cr(III)-gallate [Cr(III)-GA] and Cr(III)-ethyl gallate [Cr(III)-EGA] as final products. These Cr(III)-tannin complexes are DNA binders. The interaction of calf-thymus DNA with Cr(III)-GA and Cr(III)-EGA in aqueous solutions at physiological pH were studied at Cr(III)/DNA (phosphate) molar ratios (r) of 1 : 160, 1 : 80, 1 : 40, 1 : 20, 1 : 10, 1 : 4, and 1 : 2 using FTIR spectroscopy and capillary electrophoresis. An analysis by FTIR showed that at low concentrations (r = 1/80 and 1/40), Cr(III)-GA and Cr(III)–EGA mainly bind to the guanine N-7 atom of the G–C base pairs with minor perturbations of the A–T base pairs. At r > 1/20, a partial helix opening occurred with major perturbations of the G–C and A–T base pairs. At r > 1/10, aggregation of the Cr(III)-tannin–DNA complexes occurred. However, no DNA conformational changes were observed, and the DNA maintained the B-family structure. The binding constants of the Cr(III)-GA–DNA and Cr(III)-EGA–DNA were estimated to be 3.8 × 104 M−1 and 6.2 × 104 M−1, respectively, by Scatchard plots following capillary electrophoresis. These results suggest that the Cr(III)-tannin complexes are external DNA binders, and do not form a chelate with the DNA.

Monomer/Dimer ratios of replication protein modulate the DNA strand-opening in a replication origin

DNA opening is an essential step in the initiation of replication via the Cairns mode of replication. The opening reaction was investigated in a γ ori system by using hyperactive variants of plasmid R6K-encoded initiator protein, π. Reactivity to KMnO 4 (indicative of opening) within γ ori DNA occurred in both strands of a superhelical template upon the combined addition of wt π, DnaA and integration host factor (IHF), each protein known to specifically bind γ ori. IHF, examined singly, enhanced reactivity to KMnO 4. The IHF-dependent reactive residues, however, are distinct from those dependent on π (wt and hyperactive variants). Remarkably, the DNA helix opening does not require IHF and/or DnaA when hyperactive variants of π were used instead of wt protein. We present three lines of evidence consistent with the hypothesis that DNA strand separation is facilitated by π monomers despite the fact that both monomers and dimers of the protein can bind to iterons (π binding sites). Taken together, our data suggest that π elicits its ability to modulate plasmid copy number at the DNA helix-opening step.

Sequence-Specific Targeting of Duplex DNA by Peptide Nucleic Acids via Triplex Strand Invasion

Because of a set of exceptional chemical, physical, and biological properties, polyamide or peptide nucleic acids (PNAs) hold a distinctive position among various synthetic ligands designed for DNA-targeting purposes. Cationic pyrimidine PNAs (cpyPNAs) represent a special group of PNAs, which effectively form strand invasion triplexes with double-stranded DNA (dsDNA) also known as P-loops. Extraordinary stability of the invasion triplexes and high sequence specificity of their formation combined with local opening of the DNA double helix within the P-loops make these complexes very attractive for sequence-specific manipulation with dsDNA. Important for applications is the fact that the discrimination between correct and mismatched binding sites in dsDNA by cpyPNAs is a nonequilibrium, kinetically controlled process. Therefore, a careful choice of experimental conditions that are optimal for the kinetic discrimination of correct versus mismatched cpyPNA binding is crucial for sequence-specific recognition of dsDNA by cpyPNAs. The experimental and theoretical data presented make it possible to select those solution parameters and cpyPNA constructions that are most favorable for sequence specificity without compromising the affinity of dsDNA targeting.

Quinoxaline antibiotics enhance peptide nucleic acid binding to double-stranded DNA.

The effects of a wide range of DNA binding drugs on peptide nucleic acid (PNA) binding to double-stranded DNA by strand displacement have been investigated using a gel retardation assay. The bis-PNA [H-(Lys)-TTJTTJTTTT-(eg)(3)-TTTTCTTCTT-Lys-NH(2)] was used together with a 248 bp DNA fragment containing an appropriate target for the PNA. Most of the ligands that were studied, including DNA minor groove binders as well as intercalators and bis-intercalators, either have no effect or strongly inhibit PNA binding to DNA. By contrast, quinoxaline antibiotics facilitate PNA-DNA complex formation. The "PNA-helper" effect of echinomycin was studied in more detail using time and temperature dependence experiments to elucidate the mechanism. PNA binding to DNA follows pseudo-first-order kinetics, but the initial rate of binding is accelerated more than 10-fold in the presence of 10 microM echinomycin. The activation energy for PNA binding to dsDNA is lowered 2-fold by the antibiotic (45 vs 90 kJ/mol in the control). The reasons why quinoxalines promote the binding of PNA to DNA are not entirely clear but may well include distortions (opening) of the double helix that facilitate PNA invasion. This study establishes that the efficacy of DNA-targeted PNA antigene molecules could potentially be enhanced by judiciously adding certain DNA-interactive ligands.

Mechanical opening of the DNA double helix

Mechanical opening of the DNA double helix

Structural Analysis of DNA-Chlorophyll Complexes by Fourier Transform Infrared Difference Spectroscopy

Porphyrins and metalloporphyrins are strong DNA binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. This study was designed to examine the interaction of calf thymus DNA with chlorophyll a (CHL) in aqueous solution at physiological pH with CHL/DNA(phosphate) ratios ( r) of 1/160, 1/80, 1/40, 1/20, 1/10, and 1/5. Fourier transform infrared (FTIR) difference spectroscopy was used to characterize the nature of DNA-pigment interactions and to establish correlations between spectral changes and the CHL binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of DNA-CHL complexes in aqueous solution. Spectroscopic results showed that CHL is an external DNA binder with no affinity for DNA intercalation. At low pigment concentration ( r = 1/160, 1/80, and 1/40), there are two major binding sites for CHL on DNA duplex: 1) Mg-PO 2 and 2) Mg-N7 (guanine) with an overall binding constant of K = 1.13 × 10 4 M −1. The pigment distributions are 60% with the backbone PO 2 group and 20% with the G-C base pairs. The chlorophyll interaction is associated with a major reduction of B-DNA structure in favor of A-DNA. At high chlorophyll content ( r = 1/10), helix opening occurs, with major spectral alterations of the G-C and A-T bases. At high chlorophyll concentration (1/5), pigment aggregation is observed, which does not favor CHL-DNA complexation.

High Base Pair Opening Rates in Tracts of GC Base Pairs

Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.

DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.

Mechanical separation of the complementary strands of DNA.

We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage lambda DNA. The 3' and 5' extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10-15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100-500 bases are presently detected.