B-form Of DNA Research Articles

A comparative study on the interaction of phenazinium dyes with low pH induced protonated structure and B-form structure of naturally occurring deoxyribonucleic acid

The interaction of two phenazinium dyes namely Phenosafranine (PSF) and Safranin T (ST) with right-handed B-form and left-handed protonated form of Calf Thymus (CT) DNA was investigated using different spectroscopic techniques. Both the dyes have been shown to bind strongly to the right-handed B-form of DNA by the mechanism intercalation as revealed from fluorescence quenching, circular dichroism (CD) and viscosity measurement. From circular dichroic studies it was evidenced that both of them convert the low pH induced left-handed protonated form of DNA back to the bound right-handed form. Scatchard analysis showed that both the dyes bound strongly to B-form of DNA in a non-cooperative manner. In case of protonated form, there was sequential conversion of the polynucleotide from left-handed to the bound right-handed conformation. Our results suggest that the binding environment of the dyes in the two forms of DNA is similar and our data predict that PSF is more effective in the conversion than ST. Experimental data enabled the calculation of the number of base pairs of protonated-form that adopted a right-handed conformation for each bound dye. Our data revealed that PSF is more effective in the conversion compard to that of ST. These results are attributed to greater steric crowd in ST compared to PSF which restricts the former to intercalate between DNA base pairs. The results of these studies allow a better understanding of dye-polymorphic nucleic acid interactions at a molecular level.

Spectroscopic and molecular docking studies on the interaction of troxerutin with DNA

Troxerutin (TXER) is a derivative of naturally occurring bioflavonoid rutin. It possesses different biological activities in rising clinical world. The biological activity possessed by most of the drugs mainly targets on macromolecules. Hence, in the current study we have examined the interaction mechanism of TXER with calf thymus DNA (CT-DNA) by using various spectroscopic methods, isothermal titration calorimetry (ITC) and molecular docking studies. Further, DNA cleavage study was carried out to find the DNA protection activity of TXER. UV-absorption and emission spectroscopy showed low binding constant values via groove binding. Circular dichroism study indicates that TXER does not modify native B-form of DNA, and it retains the native B-conformation. Furthermore, no effective positive potential peak shift was observed in TXER–DNA complex during electrochemical analysis by which it represents an interaction of TXER with DNA through groove binding. Molecular docking study showed thymine guanine based interaction with docking score −7.09kcal/mol. This result was compared to experimental ITC value. The DNA cleavage study illustrates that TXER does not cause any DNA damage as well as TXER showed DNA protection against hydroxyl radical induced DNA damage. From this study, we conclude that TXER interacts with DNA by fashion of groove binding.

Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures.

Ionic mobility in DNA films studied by dielectric spectroscopy.

Double-helix DNA molecules can be found under different conformational structures driven by ionic and hydration surroundings. Usually, only the B-form of DNA, which is the only form stable in aqueous solution, can be studied by dielectric measurements. Here, the dielectric responses of DNA molecules in the A- and B-form, oriented co-linearly within fibres assembled in a film have been analyzed. The dielectric dispersion, permittivity and dissipation factor, have been measured as a function of frequency, strength voltage, time, temperature and nature of the counter-ions. Besides a high electrode polarization component, two relaxation peaks have been observed and fitted by two Cole-Cole relaxation terms. In the frequency range that we investigated (0.1 Hz to 5 ·10(6) Hz) the dielectric properties are dominated by the mobility and diffusivity of the counter-ions and their interactions with the DNA molecules, which can therefore be characterized for the A- and B-forms of DNA.

Scoliosis of backbone in Z-DNA nucleotides

According to Wikipedia "scoliosis is a medical condition in which a person's spine is curved from side to side. Although it is a complex three-dimensional deformity, on an X-ray, viewed from the rear, the spine of an individual with scoliosis can resemble an `S' or a `?', rather than a straight line". This definition fits very well with the syndrome observed in many structures of Z-DNA nucleotides. In contrast to the A- and B-forms of DNA, where the sugar-phosphate backbone smoothly follows the right-handed helical line, in Z-DNA the backbone winds as a left-handed zig-zag pattern, repeating every two base pairs. On the basis of the first structures of Z-DNA it was observed that the phosphate groups can be rotated towards the outside of the helix in the ZI type or towards the inside in the ZII type of Z-DNA. A large number of crystal structures of Z-DNA oligomers are currently available in the Protein Data Bank, where both backbone types are observed, often co-existing as alternative, partially occupied conformations. At first it was postulated that the backbone conformation adopted by Z-DNA depends on the presence of metal cations and polyamines in the crystal structures, but later it was realized that the ZI and ZII conformations are not specific to the sequence or the interactions with metal ions [1]. A comparison of larger number of high-resolution Z-DNA structures reveals that the structures can adopt a range of backbone torsion angles between the two values representing the canonical ZI and ZII types [2].

CD Spectral Studies of Pb<sup>2+</sup> Interference with 5-Fluorouracil Induced DNA Structural Changes

2+ ) a common pollutant and known toxicant, and other various anti cancer drugs such as 5-fluorouracil bind to DNA competitively. Their efficacy as anti cancer drugs was brought by their mechanism of involvement with DNA and their interactive geometries. In this perspective we have designed CD spectral studies to investigate the interference of Pb 2+ with DNA-Drug interaction geometries. The change in elliptical intensities at around 260 nm to 280 nm and 245 nm shows the conformational changes in B-form of DNA. Amplitude changes observed with in B-form indicates the hairpin formation which is an intra-molecular variant of the B-form. Calf thymus DNA with 42% of G-C content has tendency towards hairpin transitions. In this study we have found that hairpin transitions might be involved in mechanism of Pb 2+

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential

BackgroundAllicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level. MethodsMTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction. ResultsAllicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2×104M-1 for DNA and 1.06×103M−1for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin. ConclusionsThe results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin. General significanceThe results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.

Impact of Geometry Optimization on Base–Base Stacking Interaction Energies in the Canonical A- and B-Forms of DNA

Base stacking is known to make an important contribution to the stability of DNA and RNA, and accordingly, significant efforts are ongoing to calculate stacking energies using ab initio quantum mechanical methods. To date, impressive improvements have been made in the model chemistries used to perform stacking energy calculations, including extensions that include robust treatments of electron correlation with extended basis sets, as required to treat interactions where dispersion makes a significant contribution. However, those efforts typically use rigid monomer geometries when calculating the interaction energies. To overcome this, in the present work, we describe a novel internal coordinate definition that allows the relative, intermolecular orientation of stacked base monomers to be constrained during geometry optimizations while allowing full optimization of the intramolecular degrees of freedom. Use of the novel reference frame to calculate the impact of full geometry optimization versus constraining the bases to be planar on base monomer stacking energies, combined with density-fitted, spin-component scaling MP2 treatment of electron correlation, shows that full optimization makes the average stacking energy more favorable by -3.4 and -1.5 kcal/mol for the canonical A and B conformations of the 16 5' to 3' base stacked monomers. Thus, treatment of geometry optimization impacts the stacking energies to an extent similar to or greater than the impact of current state of the art increases in the rigor of the model chemistry itself used to treat base stacking. Results also indicate that stacking favors the B-form of DNA, though the average difference versus the A-form decreases from -2.6 to -0.6 kcal/mol when the intramolecular geometry is allowed to fully relax. However, stacking involving cytosine is shown to favor the A-form of DNA, with that contribution generally larger in the fully optimized bases. The present results show the importance of allowing geometry optimization, as well as properly treating the appropriate model chemistry, in studies of nucleic acid base stacking.

Optimization of the CHARMM additive force field for DNA: Improved treatment of the BI/BII conformational equilibrium.

The B-form of DNA can populate two different backbone conformations: BI and BII, defined by the difference between the torsion angles ε and ζ (BI = ε-ζ < 0 and BII = ε-ζ > 0). BI is the most populated state, but the population of the BII state, which is sequence dependent, is significant and accumulating evidence shows that BII affects the overall structure of DNA, and thus influences protein-DNA recognition. This work presents a reparametrization of the CHARMM27 additive nucleic acid force field to increase the sampling of the BII form in MD simulations of DNA. In addition, minor modifications of sugar puckering were introduced to facilitate sampling of the A form of DNA under the appropriate environmental conditions. Parameter optimization was guided by quantum mechanical data on model compounds, followed by calculations on several DNA duplexes in the condensed phase. The selected optimized parameters were then validated against a number of DNA duplexes, with the most extensive tests performed on the EcoRI dodecamer, including comparative calculations using the Amber Parm99bsc0 force field. The new CHARMM model better reproduces experimentally observed sampling of the BII conformation, including sampling as a function of sequence. In addition, the model reproduces the A form of the 1ZF1 duplex in 75 % ethanol, and yields a stable Z-DNA conformation of duplex (GTACGTAC) in its crystal environment. The resulting model, in combination with a recent reoptimization of the CHARMM27 force field for RNA, will be referred to as CHARMM36.

Interactions of Metal Ions with DNA, Its Constituents and Derivatives, which may be Relevant for Anticancer Research

In this review several types of interactions between metal ions and DNA are given, starting from basic binding to the use of metal complexes in cancer treatment and diagnostics. Metal cations help to neutralize the negative charge of DNA and thus enable the normal functions of DNA but many other interactions are also possible and are discussed in this paper. Various consequences of such interactions can be reversible (e. g. conformational changes) or irreversible (e. g. cleavage). It is known that some metal ions can also damage DNA which can provoke mutations and in some cases leads to cancer. It is clear that we know a lot about metal-DNA interactions but much more information is needed to understand the role of metal ions completely and to use this knowledge successfully.

Abstract 4243: HMGA1: A driver of the stem cell phenotype in intestinal cyrpt cells and colon cancer

Abstract Although the HMGA1 gene is highly expressed in human embryonic stem cells and high-grade, poorly differentiated cancers, its function in these settings has not been clearly elucidated. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which are members of a family of non-histone, chromatin remodeling proteins. These proteins bind to the minor groove of AT-rich regions in the B-form of DNA, which leads to the recruitment of transcriptional factors that modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis and in aggressive human cancers arising from different embryologic origins, including cancers of hematopoietic, lung, breast, prostate, and colon cells. Forced overexpression of HMGA1a or HMGA1b induces a transformed phenotype in cultured cells and inhibiting HMGA1 blocks transformation in human cancer cell lines. Because HMGA1 functions in transcriptional regulation, we hypothesize that it induces neoplastic transformation by dysregulating specific molecular pathways. To investigate the pathways disrupted by HMGA1, we are studying our HMGA1a transgenic mice as a model system. As previously reported, these mice develop aggressive T-cell leukemia with complete penetrance and females also develop uterine cancer. At necropsy, we also discovered polyps involving the small and large intestines. Histopathologically, the transgenic intestines are characterized by a thickened mucosal surface, hyperproliferative changes, and hamartomatous polyps. Immunohistochemical analysis reveals an increase in the population of intestinal cells expressing the colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr-5) in transgenic compared to wildtype mice. These data support our observation of an expansion in the crypt progenitor/intestinal stem cell populations. Our preliminary results also show that inhibiting HMGA1 expression in colon cancer cell lines interferes with anchorage-independent cell growth and tumorigenesis, indicating that HMGA1 is required for these transformation phenotypes in colon cancer. In addition, we found that HMGA1 is enriched in &amp;gt;70% of primary tumors with high-grade colon cancer. These findings suggest that HMGA1 is important in maintaining the stem cell niche in the intestine. Moreover, HMGA1 could promote neoplastic transformation and tumor progression in the intestine by driving a stem-like phenotype in colonic cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4243.

Differential stability of DNA crossovers in solution mediated by divalent cations

The assembly of DNA duplexes into higher-order structures plays a major role in many vital cellular functions such as recombination, chromatin packaging and gene regulation. However, little is currently known about the molecular structure and stability of direct DNA–DNA interactions that are required for such functions. In nature, DNA helices minimize electrostatic repulsion between double helices in several ways. Within crystals, B-DNA forms either right-handed crossovers by groove–backbone interaction or left-handed crossovers by groove–groove juxtaposition. We evaluated the stability of such crossovers at various ionic concentrations using large-scale atomistic molecular dynamics simulations. Our results show that right-handed DNA crossovers are thermodynamically stable in solution in the presence of divalent cations. Attractive forces at short-range stabilize such crossover structures with inter-axial separation of helices less than 20 Å. Right-handed crossovers, however, dissociate swiftly in the presence of monovalent ions only. Surprisingly, left-handed crossovers, assembled by sequence-independent juxtaposition of the helices, appear unstable even at the highest concentration of Mg2+studied here. Our study provides new molecular insights into chiral association of DNA duplexes and highlights the unique role divalent cations play in differential stabilization of crossover structures. These results may serve as a rational basis to understand the role DNA crossovers play in biological processes.

Antirestriction proteins ArdA and Ocr as efficient inhibitors of type I restriction-modification enzymes

Genes encoding antirestriction proteins (antirestrictases, inasmuch as the antirestriction proteins inhibit the activity of restriction-modification systems, but have no proper enzyme activity, the name antirestrictase is only tentative) are included in the composition of conjugative plasmids (genes ardABC) and some bacteriophages (genes ocr and darA). Antirestriction proteins inhibit of the type I restriction-modification enzymes and thus protect unmodified DNA of plasmids and bacteriophages from degradation. Antirestriction proteins belong to the "protein mimicry of DNA" family: the spatial structure is like the B-form of DNA, and therefore the antirestriction proteins operated on the principle of concurrent inhibition replacing DNA in the complex with the restriction-modification enzyme. Based on the prepared in vitro mutant forms of ArdA and Ocr, and also on natural proteins ArdA selectively inhibiting restriction activity of the type I enzymes, but not affecting their methylase activity, we have developed a model of complex formation between the antirestriction proteins and the restriction-modification enzymes R2M2S. Antirestriction proteins are capable of competing displacement of the DNA strand from two sites which are situated as follows: 1) in S-subunit (enzyme contact with the specific DNA site) and 2) in R-subunit (through this unit translocation of the DNA strand occurs followed by its degradation). Analysis of estriction and antimodification activities of proteins ArdA and Ocr depending on the expression level of genes ardA and ocr was performed (the cloning of the genes was done under strictly regulated promoter).

Circular Dichroism Study of the Mechanism of Formation of DNA Templated Nanowires

In order to control the fabrication method, the mechanism used in the formation of DNA templated nanowires is investigated through circular dichroism (CD) spectroscopy. Metallic (Au) and magnetic (Fe(2)O(3) and CoFe(2)O(4)) nanoparticles (NP) are aligned along the DNA strand at various mass ratios. The DNA templated nanowires are compared to the structure of B-form dsDNA through CD experiments. Absorbance and thermal melting tests are performed to verify the structural changes of DNA templated nanowires. Low concentrations of nanoparticles preserve the DNA B-form through electrostatic interactions. Conversely, at higher concentrations of nanoparticles aligned along the DNA strand, the template is denatured. Information on the mode of nanoparticle binding and DNA helix alterations are explored for metallic and magnetic nanowires based upon the results.

Ultrafast energy transfer from 3-mercaptopropionic acid-capped CdSe/ZnS QDs to dye-labelled DNA

Femtosecond-resolved fluorescence upconversion and picosecond-resolved spectroscopic measurements have been employed to confirm a highly efficient ultrafast FRET from MPA-capped CdSe/ZnS QDs to dye molecules attached to dodecamer DNA. It appears that hydrogen bonding is the associative mechanism between the MPA-capped QDs and DNA. High FRET efficiency of 92% together with the estimated donor–acceptor distance suggests that the adsorptive interactions between DNA and MPA-capped QDs result in a conformation in which DNA lies along the surface of the QD. Circular dichroism studies have been performed which reveal some perturbation in the native B-form of DNA in the nanobioconjugate.

The post-SCF quantum chemistry characteristics of the energetic heterogeneity of stacked guanine–guanine pairs found in B-DNA and A-DNA crystals

The energies of homo-guanine pairs (5′G/G-3′) in stacked conformations found in crystallographic B-DNA and A-DNA were estimated by means of DF-MP2/aDZ method with inclusion of the correction for basis superposition error. The significant heterogeneity was noticed related to the structural properties, the intermolecular interaction energies, the values of the ionization potential and the localization of the HOMO densities. The direct consequence of polymorphism of DNA is the variation of only two structural parameters defining guanine–guanine stacking. These two distinguishing variables are slide and roll. The rest of base step parameters exhibit similar variability for both A-DNA and B-DNA helices. Besides, the polymorphism of nucleic acid directly influences the stabilization of the guanine stacked pairs favoring slightly attractions in B-DNA. The most frequently occurring intermolecular interaction energies correspond to −4.87 kcal/mol and −4.20 kcal/mol for B-DNA and A-DNA forms, respectively. On the contrary, there are no significant differences in electro-donating properties of G/G pairs found in A-DNA and B-DNA since the most frequently occurring structures are characterized by almost identical IP values for both studied polymorphic forms. Most of G/G stacked complexes (>85%) are characterized by IP values belonging to the interval from 7.6 to 7.8 eV. Finally, the localization of the HOMO densities on guanine pairs was analyzed. Interestingly, irrespectively of the polymorphic form of DNA all three possible situation may take place. One may find such G/G pairs for which HOMO is located mostly on 5′-G, on 3′-G or spread out over both guanine molecules in stacked complex. The N 7 centre of 3′-G is supposed to be the source of such diversity.

Ion Dynamics and Water Percolation Effects in DNA Polymorphism

The dynamics of ions and water at the surface of DNA are studied by computer simulations in a wide range of hydrations involving the zone of low-hydration polymorphism in DNA. The long-range mobility of ions exhibits a stepwise increase at three distinct hydration levels. The first of them is close to the midpoint of the water percolation transition as well as the midpoint of the transition between A- and B-DNA forms. It coincides with the onset of the dissociation of ion pairs on the DNA surface probably caused by the increase in the water dielectric permittivity due to the appearance of the spanning hydrogen-bonding network. The other two steps are attributed to the formation of percolating water layers on the surface of DNA accompanied by the progressive escape of ions from the DNA surface. The results agree with earlier experimental data and further corroborate the suggested universal mechanism of the low hydration polymorphism in DNA including intraduplex electrostatic condensation close to the water percolation threshold.

Poly(dG)–poly(dC) DNA appears shorter than poly(dA)–poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: ∼2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)–poly(dC)], ∼2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)–poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)–poly(dC) is ∼30% shorter than that of poly(dA)–poly(dT) and the plasmid. This led us to suggest that individual poly(dG)–poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)–poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.

Crystal Structure of Bacteriophage T4 5′ Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.

The 26th International Symposium of Sapporo Cancer Seminar: Innate immunity in cancer and infectious diseases

T 26th International Symposium of the Sapporo Cancer Seminar was held at Hokkaido University, Sapporo, Japan, on July 21–23, 2006. More than 120 researchers from various countries participated in the meeting (Fig. 1). The weather was fine every day, and the participants enjoyed walks around the university campus and dining at Chara Restaurant and Kirin Beer Garden. The symposium consisted of seminars presented by leading researchers from Japan and overseas in the field of innate immunity. Oral presentations were given by 12 researchers and poster presentations were given by 21 researchers. The symposium was organized by Dr Tsukasa Seya (Hokkaido University, Japan), as the chairman, and committee members Drs Misako Matsumoto, Hirofumi Sawa, Tokiyoshi Ayabe, and Roberto Cattaneo. Ms Saoko Kume was particularly involved in the organization of this meeting.