Cyclization of three satellite components of calf thymus DNA.

Cyclization of denatured and reannealed satellite components of calf thymus DNA was studied by electron microscopy. All three satellite DNA components studied (1.707g/cm-3, 1.714g/cm-3 and 1.721g/cm-3) form circular structures indicating that the sequences of the calf thymus satellite DNAs are arranged in a tandemly repetitious manner. Under appropriate annealing conditions the amount of circular structures is reproducible and practically no aggregates are formed. By comparison of cyclization experiments under defined conditions it is demonstrated that individual satellite components differ in the amount of circular structures formed during reassociation and in the distribution of linear and circular molecules. From the distribution of the contour lengths of circular molecules we conclude that the length of the repetitive sequence decreases with increasing buoyant density of the satellite components. The average lengths of the repetitive sequences calculated from electron microscopy measurements are in good agreement with those from renaturation kinetics.

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.

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.

Basic polypeptides as histone models: circular dichroism of complexes of model polypeptides with DNA.

Circular dichroism (CD) was used to study the complexes of DNA (in 0.15M NaCl) with two polypeptides considered as models of the histone molecules. CD spectra in the region of DNA absorption were studied with respect to the concentration used for annealing and to the molecular weight and composition of the DNA used. The properties of supernatants after centrifugation of aggregated complexes were examined. The effect of selectively bound antibiotics (actinomycin D and netropsin) on CD sprectra of complexes was investigated. The induced CD of proflavine molecules bound to DNA in the various complexes was also studied. It was concluded that changes in the CD spectra of DNA in complexes with the polypeptides are due to the formation of chiral superstructures, even if some conformational changes of DNA molecules themselves may also be decisive in some cases. The superstructure is affected by the composition of DNA, the role of (G + C) rich segments being particularly important.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[Design and synthesis of peptides capable of specific binding to DNA]. / Konstruirovanie i sintez peptidov, sposobnykh spetsificheski sviazyvat'sia s DNK.

In the present communication, design, synthesis and DNA binding activities of the following two peptides are reported: Dns-Gly-Ala-Gln-Lys-Leu-Ala-Cly-Lys-Val-Gly-Thr-Lys-Val-Lys-Val-Gl y-Thr-Lys-Thr - Val-OH (I) and [(H-Ala-Lys-Leu-Ala-Thr-Lys-Ala-Gly-Val-Lys-Gln-Gln-Ser-Ile-Gln-Leu-Ile- Thr- Ala-Aca-Lys-Aca)2Lys-Aca]2Lys-Val-OH (II), where Aca = NH(CH2)5CO--; Dns is a residue of 5-dimethylaminonaphtalene-1-sulfonic acid. Peptide I contains a large fraction (ca.30%) of valyl and threonyl residues, which possess a high potential for beta structure formation. Peptide II contains four repeats of the amino acid sequence present in the presumed DNA binding helix-turn-helix unit of 434 Cro repressor. These four domains are linked in such a way that two domains can interact with two halves a 14 base pair long operator site on DNA. From CD studies we have found that peptide I is in a random coil conformation in the aqueous solution in the presence of 20% trifluoroethanol. By contrast, amino acid residues of peptide II assume alpha helical, beta and random coiled conformations under the same conditions. A change in the secondary structure of the two peptides upon binding to DNA is observed. The difference CD spectra obtained by subtracting the spectra of free DNA from the spectra of peptide I--DNA complexes gives rise to a beta-like pattern. The difference CD spectra obtained for complexes of peptide II with various natural and synthetic DNAs suggest that alpha-beta-transition takes place in the presumed helix-turn-helix repeat units of peptide II upon binding to DNA. Peptide I binds more strongly to poly(dG).poly(dC) than to poly(dA).poly(dT) and poly[d(GC)].poly[d(GC)]. The binding takes place in the minor DNA groove because minor groove binding antibiotic sibiromycin can displace peptide I from a complex with poly(dG).poly(dC). Analysis of footprinting diagramms shows that peptide I specifically protects phosphodiester bonds within operator sites OR1 and OR2 of phage lambda from nuclease cleavage. By contrast, peptide II does not react specifically with operators OR1, OR2 and OR3 of phage 434 although it forms very tight complexes with DNA which are stable in the presence of 1M NH4F.

Reassociation kinetics of three satellite components of calf thymus DNA.

Using absorption measurements the reassociation kinetics of three satellite DNA components isolated from calf thymus was studied under various conditions. A different method using CsC1 density gradient determinations particularly suited for kinetic analysis of mixtures was also used and shown to give similar results. Reassociation rate constants were corrected for mismatching during strand reassociation using data obtained by kinetic analysis of fractions of the 1.714 g/cm-3 satellite component. The values of corrected as well as uncorrected complexities were calculated and compared with results of other methods. They were shown to be compatible with the concept of sequence repetition at various levels.

Identification and separation of components of calf thymus DNA using a CsC1-netropsin density gradient.

Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparativeCsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm minus 3, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm minus 3 appear to be heterogeneous.