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Biomedical subjects

J Sponar

Publications and source records attributed to J Sponar.

At least 19 recordsLinked to original sources
PubMed1997

Interaction of a bZip oligopeptide model with oligodeoxyribonucleotides modelling DNA binding sites. The effect of flanking sequences.

A leucine zipper (bZip) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZip proteins (Jun) with related DNA binding specificity. Self-complementary DNA hexadecanucleotides containing ATF/CRE, AP-1 and C/EPB target sites were used to study peptide-DNA complex formation. Conformation changes in both components that occur on complex formation were studied by circular dichroism (CD) spectroscopy. The results show that the amount of alpha-helix formed in the peptide strongly depends not only on the target site present, but also on the type of the sequence flanking the ATF/CRE target site. Highest amount of the alpha-helix induced in the peptide was observed when homopurine homopyrimidine flanking sequences were present, whereas the presence of alternating sequences, especially of the CA/TG type, showed considerably lower effects. The change in DNA conformation on complex formation was generally small, but also depended on the type of the flanking sequence. It appears that the sequences flanking the target site can considerably modify the ability of the target sequence to bind specifically the bZip peptide, probably by slightly varying the overall DNA conformation.

Activating Transcription Factor 2↗
PubMed1996

Selective binding of synthetic polypeptides to DNA of varying composition and sequence: effect of minor groove binding drugs.

Repetitive basic polypeptides containing lysine or arginine as every third amino acid were shown to cause DNA condensation at physiological salt concentration connected with selective DNA binding with respect to DNA composition and sequence. This selectivity is very similar to that existing in the case of histone H1 and other basic proteins and does not depend on polypeptide chain conformation. The effect of the minor groove binding drugs netropsin and distamycin was tested to elucidate the origin of the binding selectivity. The results suggest that the binding preferences are due to the variations in the conformation in various types of B-DNA that depend on DNA composition and sequence. The most important factor affecting the selectivity is probably the value of the negative electrostatic potential in the minor groove.

Base Composition↗
PubMed1995

Protein-induced bending or flexing at the 5'-end of the duck beta A-globin promoter.

The 5'-end of the duck beta A-globin promoter contains a protein binding site BS-3/Sp1, the A + T-rich part of which could be involved in DNA bending. Plasmids were constructed using the pBend2 plasmid containing BS-3/Sp1. Circular permutation analysis of the fragments cut out from the plasmids using various restriction endonucleases, in the presence of a partially purified protein extract from embryonic duck erythrocytes, was performed. The results indicate that a rather complicated change in the fragment shape takes place upon protein binding, which is best explained as an induction of two points of bending or flexure within the fragment. Analogical points of flexure may exist at the protein-binding sites of the duck and chicken beta A-globin promoters in spite of differing DNA sequences.

Animals↗
PubMed1994

An empirical potential study of the interaction of L-lysine-L-alanine-L-alanine tripeptide with four models of B-DNA with different compositions.

The empirical potential including the intra- and intermolecular energy terms was used to study the interaction of L-Lysine-L-Alanine-L-Alanine Tripeptide with four models of B-DNA with different compositions. On the basis of a detailed search of the respective potential energy surface, it was found that the peptide is preferentially bounded to the AT-rich sequences. Analysis of the different energy contributions indicated that the electrostatic term is responsible for this preference. The results agree with the experimental data on the selectivity of some DNA--binding proteins and polypeptides to AT-rich DNA.

Base Sequence↗
PubMed1994

B-Z transition of synthetic oligonucleotides containing non-Z forming elements.

Ten oligonucleotides of the length 8-12 base pairs have been synthesized, which contain, in addition to the obligatory sequences CG/CG, sequences not favorable for the transition to the Z conformation (A.T pairs, GG/CC or AA/TT sequences). Conformational transitions of these oligonucleotides in high concentrations of NaClO4 in the absence and in the presence of Ni2+ were investigated using CD spectroscopy. The B-Z transition is affected by the length and sequence of the oligonucleotide. Increasing the NaClO4 concentration alone the transition of only one of the oligonucleotides studied. (CGCGCGTGCACGCGCG)2, can be induced. Other oligonucleotides remain in the B conformation or only partial transition to the Z conformation can be observed. Most other oligonucleotides can be converted into the Z conformation at intermediate concentrations of NaClO4 (2.0-3.2 M) by an addition of Ni2+ ions. In some cases, however, Ni2+ can destabilize the double stranded structure of the sample. We have studied the effect of the presence of A.T pairs in the G.C containing oligonucleotides and the effect of the presence of pu-pu/pyr-pyr sequences. The presence of the latter sequences in the Z form implicates the formation of a Z-Z'junction which makes the transition quite difficult. Despite the fact that some oligonucleotides contained several structural elements not favorable for the transition, we did not find any sequence which would completely block the ability of the oligonucleotide to adopt the Z conformation.

Base Composition↗
PubMed1993

B-Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of basic oligopeptides.

The effect of basic oligopeptides (Lys-Ala-Ala)n and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-m5dC).poly(dG-m5dC) in aqueous solution and in methanol-water mixtures was investigated by c.d. spectroscopy. In aqueous solution dimers and higher oligomers induce a transition to the Z conformation. These temperature dependent transitions are consistent with positive delta HB-Z values which depend on peptide composition. In the absence of peptides no transition can be observed. In the presence of peptides B-Z transition can be induced by small amount of methanol. The temperature dependence of this transition is consistent with a small, but definitely negative delta HB-Z. At high methanol concentration a transition to Z' type conformation was observed. In subcritical methanol concentrations B-Z transition can be induced by the addition of peptides. In this case the delta HB-Z values are again very small, but definitely positive. The effects of bulky hydrophobic peptide side chains, of the presence of methanol and the temperature dependences are consistent with an important contribution of hydrophobic interactions in maintaining the stability of the Z DNA-peptide complex.

Amino Acid Sequence↗
PubMed1991

Effect of basic oligopeptides on the B-Z transition of poly(dG-dC). poly(dG-dC) in water-methanol solutions.

The effect of basic oligopeptides (Lys-Ala-Ala)n (n = 1-5, 10) and (Lys-Leu-Ala)n (n = 1-4) on the B-Z transition of poly(dG-dC).poly(dG-dC) in water-methanol solutions was investigated using CD and uv spectroscopy. In the absence of peptides, the concentration of methanol at the midpoint of the B-Z transition is 64% at 25 degrees C. The transition is temperature dependent and the B conformation is preferred at higher temperatures. All peptides tested shift the midpoint of the B-Z transition to lower concentrations of methanol. For shorter peptides this effect increases with an increasing number of monomeric units, showing the importance of the number of positive charges in the peptide molecule. Al conditions of low methanol content, the trimer and tetramer of the (Lys-Leu-Ala)n series have a greater effect on the B-Z transition than the corresponding oligomers of the (Lys-Ala-Ala)n series. This indicates an important influence of the presence of hydrophobic groups in the peptide side chains on the binding. In the presence of peptides, the B-Z transition is also temperature dependent and the B conformation is preferred at higher temperatures. The addition of peptides results in an increase of the transition midpoint and of the transition width. These parameters were used for the calculation of the transition enthalpy delta HB-Z in 65% methanol, which is -1.15 +/- 0.25 kcal/base pair. Since the van't Hoff enthalpy delta HVH calculated from the temperature dependence of the B-Z transition in the absence of peptides is -130 kcal/mol, the length of the cooperative unit is about 110 base pairs. The results suggest that the mechanism of Z-DNA induction is similar but not identical with that involved in the action of metal cations in aqueous solution.

Amino Acid Sequence↗
PubMed1990

Flexibility of A.T pairs in left-handed DNA helices.

We have analysed by various approaches the structure of cloned synthetic sequences in supercoiled plasmids. Individual inserts were formed by d(C-G)n blocks interrupted by the presence of A.T pairs positioned either in phase or out of phase of pur-pyr alternation. Based on the thermodynamic analysis we obtained results confirming that A.T pairs are easily incorporated into left-handed helices without significant energetic penalty. Sequences GTAC which are known to form cruciform structures in multiple repetition underwent a B-Z transition. In the case of plasmids containing AA/TT code words and substantial discontinuities in purine-pyrimidine alternation our analysis indicates that Z-Z junctions formed by A.T pairs contributed little to the overall energetic demands of the B-Z transition probably thanks to their high conformational flexibility.

Base Composition↗
PubMed1989

Supercoil-induced Z-DNA formation within 5'-end of chicken myb proto-oncogene.

We have analyzed the recently sequenced and characterized 2.9 kb fragment derived from the 5'-end of chicken myb proto-oncogene with respect to structural perturbations induced by DNA supercoiling. Within the first intron a 50 bp sequence stretch was localized, starting approximately 450 nucleotides downstream from putative ATG initiation codon, which forms a non-B-DNA structure. Fine mapping with structural probes revealed the three adjacent regions with imperfect purine-pyrimidine alternation creating together relatively long Z-forming tract, parts of which may undergo a B-Z DNA transition at different superhelical densities.

Alkylation↗
PubMed1989

Conformational transitions of leucine-containing isomeric sequential basic polytripeptides.

Conformational transitions of basic sequential polytripeptides (Lys-Ala-Leu)n, (Arg-Ala-Ala)n, (Arg-Leu-Ala)n, and (Arg-Ala-Leu)n, induced by elevated salt concentrations and/or temperatures in aqueous solutions, were investigated by CD, sedimentation equilibrium, and viscometry. The behavior of (Lys-Ala-Leu)n was compared with that of the sequential isomer (Lys-Leu-Ala)n, studied previously. It was found that both polypeptides are highly helical with a tendency to aggregate in high salt solutions. Although the hydrophobic interactions between Lys and Leu residues play an important role in both cases, the final effect on helix stabilization and aggregation is different. The Arg-containing polypeptides were found to assume the alpha-helical conformation. Compared to the Lys-containing polypeptides (Lys-Ala-Leu)n and (Lys-Leu-Ala)n, a very low tendency to aggregate was observed.

Isomerism↗
PubMed1988

B-X transition in synthetic and natural (dA-dT)n.(dA-dT)n sequences.

AB-X transition of polyh(dA-dT).poly(dA-dT) was observed to occur in methanol-water mixtures with methanol concentrations higher than 50% in the presence of a specific combination of monovalent and divalent cations. In the presence of Na+, divalent cations induce denaturation of poly(dA-dT).poly(dA-dT) accompanied by condensation and/or aggregation, and effect similar to that observed previously with random sequence DNA (Votavová, Kucerová, Felsberg and Sponar, J. Biomol. Struct. Dyn. 4,477-489, 1986). In the presence of Cs+ cations a B-X transition was induced by addition of Ca2+ or Mn2+ but not Mg2+ or Ni2+ ions. Circular dichroism and ultraviolet spectroscopy demonstrate that the X conformation is a double stranded form of poly(dA-dT).poly(dA-dT) belonging presumably to the B family which, however has an altered base stacking. The X conformation of poly(dA-dT).poly(dA-dT) found in methanol-water mixtures is a condensed and/or aggregated form. In contrast, the X conformation characterized by similar CD spectra observed in high salt concentrations is not aggregated up to a concentration of 6 M CsF. In methanol-water mixtures (A+T)-rich bacterial DNA behaves essentially as a random sequence DNA revealing no detectable amount of the X form. On the other hand crab (Cancer pagurus) satellite and crab non-satellite DNAs containing varying amounts of (dA-dT)n.(dA-dT)n sequences were shown to undergo a B-X transition, at least partly, in both methanol-water mixtures and 6 M CsF solutions.

Animals↗
PubMed1986

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures.

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.

Animals↗
PubMed1985

Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers.

Using CD measurements we show that the interaction of netropsin to poly(dA-dT).poly(dA-dT) involves two binding modes at low ionic strength. The first and second binding modes are distinguished by a defined shift of the CD maximum and the presence of characteristic isodichroic points in the long wavelength range from 313 nm to 325 nm. The first binding mode is independent of ionic strength and is primarily determined by specific interaction to dA.dT base pairs. Employing a netropsin derivative and different salt conditions it is demonstrated that ionic contacts are essential for the second binding mode. Other alternating duplexes and natural DNA also exhibit more or less a second step in the interaction with netropsin observable at high ratio of ligand per nucleotide. The second binding mode is absent for poly(dA).poly(dT). The presence of a two-step binding mechanism is also demonstrated in the complex formation of poly(dA-dT).poly(dA-dT) with the distamycin analog consisting of pentamethylpyrrolecarboxamide. While the binding mode I of netropsin is identical with its localization in the minor groove, for binding mode II we consider two alternative interpretations.

Binding Sites↗
PubMed1982

Conformational transitions of poly(dA-dT)poly(dA-dT) in ethanolic solutions.

Examination of circular dichroic and phosphorus nuclear magnetic resonance spectra showed that poly(dA-dT)-poly(dA-dT) exhibited an ethanol-induced transition to the A form in an Na+ containing medium like natural DNAs. A mere replacement of the Na+ by Cs+ counterions meant that the polynucleotide was with a little cooperativity transformed into a novel conformation displaying a deep negative band in the long wavelength part of the CD spectrum. The presence of very low concentration of Cs2+ shifted the midpoint of the transition to a lower content of ethanol.

Circular Dichroism↗
PubMed1982

A Z-like form of poly(dA-dC).poly(dG-dT) in solution?

Circular dichroism was used to study changes in conformation of poly(dA-dC).poly(dG-dT) caused by a high concentration of various monovalent salts. It was found that CsF induced the gradual appearance of a negative band in the long wavelength part of the CD spectrum of poly(dA-dC).poly(dG-dT), which might reflect a transition of this DNA toward a Z-like structure.

Circular Dichroism↗
PubMed1981

Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences.

The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.

Amino Acid Sequence↗
PubMed1979

Changes in the circular dichroic spectrum of calf thymus solubilized chromatin caused by ultraviolet irradiation.

UV irradiation of the chromatin caused an increase of the positive circular dichroic band in the vicinity of 275 nm (corresponding to DNA) and a deepening of the negative band of proteins at about 225 nm. These changes in the circular dichroic spectrum are monotonous in the range of doses studied (less than 6 X 10(4) J.m-2). The increase of the positive circular dichroic band probably reflects the occurrence of local conformational changes in DNA, which include changes in base position (tilting, distance from helix axis) in the close neighborhood of photoproducts. The presence of photoproducts in chromatin reduces changes in its circular dichroic spectra with temperature.

Animals↗