5-Methylcytosine containing CG decamer as Z-DNA embedded sequence for a potential Z-DNA binding protein probe

Abstract

Attempting to elucidate biological significance of the left-handed Z-DNA is a research challenge due to Z-DNA potential role in many diseases. Discovery of Z-DNA binding proteins has ignited the interest in search for Z-DNA functions. Biosensor with Z-DNA forming probe can be useful to study the interaction between Z-DNA conformation and Z-DNA binding proteins. In this study, 5-methylcytosine (^mC) containing CG decamers were characterized for their suitability to form Z-DNA and to be used in Z-DNA forming probe. The 5′-thiol oligonucleotide embedded with 5′-^mCG^mCG^mCG^mCG^m CG-3′ was designed and developed as a potential Z-DNA forming probe for Z-DNA binding protein screening.     

Recognition of left-handed Z-DNA of short unmodified oligonucleotides under physiological ionic strength conditions

The left-handed Z-DNA form of the short unmodified alternating guanine-cytosine oligonucleotides, 5′-(dGdC)₂₄ and 5′-(dGdC)₁₈, was selectively detected under physiological ionic strength and pH conditions using the anionic nickel(II) porphyrin, NiTPPS. No spectroscopic signal was observed for NiTPPS with any right-handed oligonucleotides under identical conditions. The 48mer 5′-(dGdC)₂₄ Z-form was detected at concentrations as low as 100 nM. The binding of NiTPPS to the B- and Z-oligonucleotides was studied quantitatively by UV-vis absorption and circular dichroism spectroscopies. NiTPPS was found to be a universal DNA binder, with binding affinity and geometry depending on the ionic composition of the solution, rather than on the DNA helical twist. This is the first example of a successful spectroscopic detection of the Z-DNA of short unmodified oligonucleotides under physiological pH and ionic strength conditions.     

Effects of base substituents on the hydration of B- and Z-DNA: correlations to the B- to Z-DNA transition.

We present a study of how substituent groups of naturally occurring and modified nucleotide bases affect the degree of hydration of right-handed B-DNA and left-handed Z-DNA. A comparison of poly(dG-dC) and poly(dG-dm5C) titrations with the lipotropic salts of the Hofmeister series infers that the methyl stabilization of cytosines as Z-DNA is primarily a hydrophobic effect. The hydration free energies of various alternating pyrimidine-purine sequences in the two DNA conformations were calculated as solvent free energies from solvent accessible surfaces. Our analysis focused on the N2 amino group of purine bases that sits in the minor groove of the double helix. Removing this amino group from guanine to form inosine (I) destabilizes Z-DNA, while adding this group to adenines to form 2-aminoadenine (A') stabilizes Z-DNA. These predictions were tested by comparing the salt concentrations required to crystallize hexanucleotide sequences that incorporate d(CG), d(CI), d(TA) and d(TA') base pairs as Z-DNA. Combining the current results with our previous analysis of major groove substituents, we derived a thermodynamic cycle that relates the systematic addition, deletion, or substitution of each base substituent to the B- to Z-DNA transition free energy.     

Stability and properties of Z-DNA containing artificial nucleobase 2′-O-methyl-8-methyl guanosine

We synthesized several DNA oligonucleotides containing one or several 2′-O-methyl-8-methyl guanosine (m⁸Gm) and demonstrated that these oligonucleotides not only stabilize the Z-DNA with a wide range of sequences under low salt conditions but also possess high thermal stability. Using artificial nucleobase-containing oligonucleotides, we studied the interaction of the Zα domain with Z-DNA. Furthermore, we showed that the m⁸Gm-contained oligonucleotides allow to study the photochemical reaction of Z-DNA.     

Chemical detection of Z-DNA within the maize Adh1 promoter

Z-DNA is a left-handed helix which can form within tracts of alternating purines and pyrimidines. Tracts of potential Z-DNA identified by sequence inspection are often noted within regulatory portions of genes, but evidence that these tracts of sequence actually exist as Z-DNA is very limited, and not available for any plant gene. In this study, the chemical probes osmium tetroxide, diethylpyrocarbonate and hydroxylamine were used to show that a tract of alternating purines and pyrimidines in the Adh1 promoter (from -311 to -325) actually assumes a Z-DNA conformation under superhelical stress in vitro.     

The conformational effect of para-substituted C8-arylguanine adducts on the B/Z-DNA equilibrium

The B form of DNA exists in equilibrium with the Z form and is mainly affected by sequence, electrostatic interactions, and steric effects. C8-purine substitution shifts the equilibrium toward the Z form though how this interaction overcomes the unfavorable electrostatic interactions and decrease in stacking in the Z form has not been determined. Here, a series of C8-arylguanine derivatives, bearing a para-substituent were prepared and the B/Z equilibrium determined. B/Z ratios were measured by CD and conformational effects of the aryl substitution determined by NMR spectroscopy and molecular modeling. The para-substituent was found to have a significant effect on the B/Z DNA equilibrium caused by altering base-pair stacking of the B form and modifying the hydration/ion shell of the B form. A unique melting temperature versus salt concentration was observed and provides evidence relevant to the mechanism of B/Z conformational interconversion.     

Quantitative analysis of DNA secondary structure from solvent-accessible surfaces: the B- to Z-DNA transition as a model

Solvent structure and its interactions have been suggested to play a critical role in defining the conformation of polynucleotides and other macromolecules. In this work, we attempt to quantitate solvent effects on the well-studied conformational transition between right-handed B- and left-handed Z-DNA. The solvent-accessible surfaces of the hexamer sequences d(m5CG)3, d(CG)3, d(CA)3, and d(TA)3 were calculated in their B- and Z-DNA conformations. The difference in hydration free energies between the Z and the B conformations (delta delta GH(Z-B] was determined from these surfaces to be -0.494 kcal/mol for C-5 methylated d(CG), 0.228 kcal/mol for unmethylated d(CG), 0.756 kcal/mol for d(CA)-d(TG), and 0.896 kcal/mol for d(TA) dinucleotides. These delta delta GH(Z-B) values were compared to the experimental B- to Z-DNA transition energies of -0.56 kcal/mol that we measured for C-5 methylated d(CG), 0.69-1.30 kcal/mol reported for unmethylated d(CG), 1.32-1.48 kcal/mol reported for d(CA)-d(TG), and 2.3-2.4 kcal/mol for d(TA) dinucleotides. From this comparison, we found that the calculated delta delta GH(Z-B) of these dinucleotides could account for the previous observation that the dinucleotides were ordered as d(m5CG) greater than d(CG) greater than d(CA)-d(TG) greater than d(TA) in stability as Z-DNA. Furthermore, we predicted that one of the primary reasons for the inability of d(TA) sequences to form Z-DNA results from a decrease in exposed hydrophilic surfaces of adjacent base pairs due to the C-5 methyl group of thymine; thus, d(UA) dinucleotides should be more stable as Z-DNA than the analogous d(TA) dinucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human telomere d[(TTAGGG)4] undergoes a conformational transition to the Na+-form upon binding with sanguinarine in presence of K+

Guanine-rich telomeric sequences fold into G-quadruplex conformation and are known to bind a variety of ligands including potential drug candidates. By means of CD spectroscopy and fluorescence lifetime measurements we demonstrate that putative anticancer therapeutic sanguinarine (SGR) exhibits two distinct interactions with human telomere d[(TTAGGG)₄] (H24) in presence of K⁺. Up to about 1:2 M ratio of H24:SGR (10 μM H24), two molecules of SGR bind H24. Above this molar ratio, SGR induces a conformational transition in H24 from the K⁺-form to the Na⁺-form. The demonstration of SGR-induced conformational transition in a G-quadruplex formed by a human telomeric sequence could provide new insights into interaction of drugs with quadruplex DNA structure.     

A study on the positive ellipticity in the circular dichroism of ribonuclease A

The small positive elliplicity near 239 nm in the CD spectrum of RNase has been investigated as a function of pH. Theoretical calculations using CD parameters representing buried or exposed tyrosine residues have been carried out. A comparison of the theoretical calculations with experimental data suggests that the changes in the band's intensity, as a function of pH, arise mainly from electronic transitions associated with the tyrosine residues. The buried tyrosine residues are the major contributors to the ellipticity in this region at neutral pH. At higher pH contributions from exposed residurs are also observed.     

Increase in temperature induces the Z to B transition of poly[d(G-C)] in water-ethanol solution

An increase in temperature from 20 to 50° C results in the complete transition from the Z to B form of poly(d(G-C)], dissolved in a 55% ethanol-water solution. The transition is fully reversible and displays a slow kinetics. The transition profiles for the free polynucleotide and for that in the presence of ethidium bromide, which is known to stabilize the B form, are obtained by circular dichroism. Based on these data the enthalpy value for the B-Z transition in our conditions is estimated to be ΔH_BZ = ?0.7 $\text{kcal}\text{mol}$.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva.

Results of a study on the solution behaviour of the cell-wall polysaccharide named ulvan obtained from hot water extraction of a flour of Ulva ‘rigida’ are reported. In particular the spectroscopic properties and ion binding capacity of this charged polysaccharide were studied by circular dichroism and isothermal microcalorimetric titrations in order to gain information on the potential exploitation of this low cost biomass. A marked tendency of this polysaccharide to uptake water was evidenced by studying the proton spin-lattice relaxation times of the solvent, T₁, embedded in this highly charged polysaccharide.     

A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva

Results of a study on the solution behaviour of the cell-wall polysaccharide named ulvan obtained from hot water extraction of a flour of Ulva ‘rigida’ are reported. In particular the spectroscopic properties and ion binding capacity of this charged polysaccharide were studied by circular dichroism and isothermal microcalorimetric titrations in order to gain information on the potential exploitation of this low cost biomass. A marked tendency of this polysaccharide to uptake water was evidenced by studying the proton spin-lattice relaxation times of the solvent, T₁, embedded in this highly charged polysaccharide.     

The role of methylation in the intrinsic dynamics of B- and Z-DNA

Methylation of cytosine at the 5-carbon position (5 mC) is observed in both prokaryotes and eukaryotes. In humans, DNA methylation at CpG sites plays an important role in gene regulation and has been implicated in development, gene silencing, and cancer. In addition, the CpG dinucleotide is a known hot spot for pathologic mutations genome-wide. CpG tracts may adopt left-handed Z-DNA conformations, which have also been implicated in gene regulation and genomic instability. Methylation facilitates this B-Z transition but the underlying mechanism remains unclear. Herein, four structural models of the dinucleotide d(GC)(5) repeat sequence in B-, methylated B-, Z-, and methylated Z-DNA forms were constructed and an aggregate 100 nanoseconds of molecular dynamics simulations in explicit solvent under physiological conditions was performed for each model. Both unmethylated and methylated B-DNA were found to be more flexible than Z-DNA. However, methylation significantly destabilized the BII, relative to the BI, state through the Gp5mC steps. In addition, methylation decreased the free energy difference between B- and Z-DNA. Comparisons of α/γ backbone torsional angles showed that torsional states changed marginally upon methylation for B-DNA, and Z-DNA. Methylation-induced conformational changes and lower energy differences may contribute to the transition to Z-DNA by methylated, over unmethylated, B-DNA and may be a contributing factor to biological function.     

Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate

The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Empirical validation of a two-state kinetic model for the B-Z transition of double-stranded poly[d(G-m5C)]

5 C)] in terms of the salt concentration, the absolute temperature, and the cooperativity index. The validity of the formula was tested using circular dichroism spectroscopy after variation of the type of salt (NaCl, MgCl₂), the salt concentration, and the temperature of the polynucleotide solution. A consequence of the proposed function is that in conditions of high salt there is a predictable salt threshold which determines the particular molecular mechanism of the B-Z transition. The paper also describes the way in which this threshold level is temperature dependent. A detailed comparison of our data with the experimental data found by other authors is given. The function agrees quantitatively with the experiments and explains the contrasting results found in the literature about the influence in the B-Z transition of both the temperature and the polymer size. Received: 21 April 1999 / Accepted: 9 September 1999     

Contributions of tryptophan side chains to the far-ultraviolet circular dichroism of proteins

It has often been assumed that the role of aromatic side chains in the far-ultraviolet region of protein circular dichroism (CD) is negligible. However, some proteins have positive CD bands in the 220–230 nm region which are almost certainly due to aromatic side chains. The contributions to the CD of interactions between tryptophan side chains and the nearest neighbor peptide groups have been studied, focusing on the indole B_b transition which occurs near 220 nm. Calculations on idealized peptide conformations show that the CD depends strongly on both backbone and side-chain conformation. Because of the low symmetry of indole, rotation about the CC bond (dihedral angle ₂) by 180° generally leads to large changes in the CD, often causing the B_b band to reverse sign. When side-chain conformational preferences are taken into account, there is no strong bias for either positive or negative B_b rotational strengths. The observation that simple tryptophan derivatives such as N-acetyl-L-tryptophan methylamide have positive CD near 220 nm implies either that these derivatives prefer the _R region over the region, or that there is little preference for ₂ < 180° over ₂ > 180°. Nearest-neighbor-only calculations on individual tryptophans in 15 globular proteins also reveal a small bias toward positive B_b bands. Rotational strengths of the B_b transition for some conformations can be as large as 1.0 Debye-Bohr magnetons in magnitude, corresponding to maximum molar ellipticities greater than 10⁵ degcm²/dmol. Although a substantial amount of cancellation occurs in most of the examples considered here, such CD contributions could be significant, especially in proteins of low helix content.     

The identification of a proton pump on vacuoles of the yeast Saccharomyces carlsbergensis. ATPase is electrogenic H+-translocase

A cadmium-binding protein rich in cysteine and acidic amino acid residues was isolated from roots of Agrostis gigantea. The molar ratio of cysteine to cadmium was 2.7:1. Electronic absorption and circular dichroism measurements were characteristic of cadmium-thiolate coordination. The cadmium-binding centre in the plant protein was strikingly similar to that of the well characterised vertebrate cadmiumthioneins.