Temperature mediated variation of DNA secondary structure in (A.T) clusters; evidence by use of the oligopeptide netropsin as a structural probe.

The titration viscometric investigation of the multi-mode interaction of netropsin (Nt) with (A.T) clusters of NaDNA12 and NH4DNA10 has been extended to different temperatures. The position of two boundaries on the r-scale (r= [Nt]bound/[DNA-P]) with increasing temperature steadily (rI/II) or more abruptly (rO/I) shifts to lower values. For the most (A.T) rich Nt-binding sites of modes (O), (I) and (II) this observation suggests the existence of an equilibrium between different DNA secondary structures with a different translation per base pair. The mode specific changes delta L1Nt of DNA contour length as induced by one Nt molecule proved to be almost independent of temperature. Concomitant stiffening effects increase with decreasing temperature, contrary to initial expectation. Conformational variability of (A.T) clusters may represent an essential feature in specific or selective DNA-protein interaction.     

Counterion-Type Characteristic Effects on Intrinsic Bending Components of Calf Thymus DNA; Hydrodynamic Investigations

Abstract

This paper stresses structural differences in A · T clusters of the ammonium salt of calf thymus (et) DNA (ctNH₄DNA) and the respective sodium salt, ctNaDNA Sequence mediated intrinsic helix bends of ctNaDNA distributed along the molecule partially randomly and partially phased with the helix screw (accompanying paper), are enhanced in ctNH₄DNA. Additionally, the number of the most strongly bent segments (of A-tract character) is raised in ctNH₄NA by a counterion mediated shift of the equilibrium between at least two local DNA conformations. Nevertheless, the apparent DNA elongation, induced by the abolition of a single apparent solenoid-related DNA tertiary structure component which generates a special intrinsic DNA bend, is the same for NH₄DNA and NaDNA.

These conclusions follow from two independent sets of experimental results:

(1.) Titration viscometric measurements with ctNH₄DNA as a function of the cation concentration in comparison to ctNaDNA (KER et al. JBSD 9, 537 (1991)) and respective DNA conformational analyses.

(2.) Quantitative viscometric analysis of DNA conformational changes on netropsin (Nt) interaction of ctNHjDNA at different temperatures and comparison with the respective data for ctNaDNA (KER et al., NAR 9, 2335 (1981).     

Interactions of spermidine and methylspermidine with DNA studied by nuclear magnetic resonance self-diffusion measurements.

The NMR pulsed field gradient self-diffusion method has been used to study the self-diffusion of the polyamine spermidine and the polyamine analog methylspermidine (completely N-methylated spermidine). The self-diffusion coefficient, D, was measured in solutions of calf thymus DNA prepared from nucleosome core particles (with an average length of 120 base pairs) as a function of the concentration ratio of polyamine to DNA phosphate. A study of the self-diffusion quotient, D/Do (where Do is the diffusion coefficient for free polyamine, not associated with DNA), in additions of spermidine and methyl-spermidine to solutions of NaDNA/NaCl, gave almost identical results with complete association of polyamine to DNA in the initial part of the titrations, indicating similar affinities for DNA. A large influence on the measured self-diffusion coefficients was detected for methylspermidine in NaDNA solutions with different concentrations of NaCl, which shows a considerable salt effect on the polyamine-DNA association. No notable differences in D/Do for methylspermidine were observed in competitive titrations of solutions of Li- and NaDNA, indicating that sodium and lithium ions behave similarly in their interactions with DNA. In titration experiments of methylspermidine into MgDNA solution, the results showed that the polyamine association is less effective than in the case of NaDNA, because of competition from magnesium binding to DNA. Comparisons with calculations based on the electrostatic Poisson-Boltzmann cell model were performed. It is suggested that the interaction is primarily of electrostatic nature, with no binding to specific sites on the DNA molecule.     

Abolition of Intrinsically Bent DNA Structure Components in AT Clusters by Netropsin Interaction; Titration Viscometric Investigations

Abstract

It is argued that the enhancement of the apparent DNA contour length by the specifically binding non-intercalating drug netropsin (Nt) (Reinert et al., NAR 9,2335, 1981) at very low Nt/DNA-phosphate ratios essentially is the result of an abolition of periodically arranged intrinsic helix bends in A · T rich tracts of base pairs.

In the preceding paper the existence of pronounced DNA tertiary structure components has been postulated for (two species of) natural eukaryotic DNA. The resulting model suggests local apparent solenoid-related DNA tertiary structure components at high sodium ion concentration c_s, partly/totally molten out at 45/60 C. With decreasing c_s the tertiary structure components have been found to be gradually reduced, at least below c_s = 0.010 M, as titration viscometrically revealed by a gradual rise of the apparent DNA contour length (Reinert et al., JBSD 9, 537, 1991).

Hence, we performed titration viscometric analyses about Nt interaction with calf thymus DNA (ctDNA) at c_s = 0.075 M, 0.010 M and 0.004 M Na⁺. The concomitant DNA conformational changes are quantitatively described in terms of the relative changes of both DNA persistence length and hydrodynamically operative apparent DNA contour length for the three first resolved interaction modes below a Nt/DNA-P ratio of 0.03.

These experiments, together with previous respective analyses at c_s = 0.20 M Na⁺ and different temperatures (I.e.), suggest that those DNA sites binding Nt most strongly predominantly are responsible for the formation of solenoid-related DNA tertiary structure components. Most probably these are A tract-containing sequences. As the essential factor for their apparent elongation effect at low Na⁺ concentrations, a gradual alteration of the number of base pairs per helix turn seems to occur below c_s = 0.010 M Na⁺ and, concomitantly, a change in phasing between intrinsic helix bends and helix screw.     

Multimode Interaction of Hoechst 33258 with Eukaryotic DNA; Quantitative Analysis of the DNA Conformational Changes

Abstract

The interaction of the minor groove binding ligand Hoechst 33258 (Hoe) with natural DNA was investigated by high resolution titration rotational viscometry. Analysis of the concomitant DNA conformational changes was performed with two DNA samples of sufficiently different molar mass M, at 4°C, 22°C and 40°C, for Hoe/DNA-P ratios below r = 0.02. In this narrow r range several interaction modes could be resolved. The measured conformational changes were quantified in terms of relative changes of both apparent DNA persistence length, Δa/a, and hydrodynamically operative DNA contour length, ΔL/L. Δa/a(r) primarily is a measure of ligand-induced DNA helix stiffening, but both, Δa/a(r) and ΔL/L(r), generally depend also on ligand binding induced DNA bending or DNA unbending. The essential difference obviously is that Δa/a(r) is influenced by the randomly distributed helix bends and ΔL/L(r) by phased ones. The measurements performed at different temperatures deliver informations about existence and temperature dependent abolition of intrinsic helix curvature.

Both Hoe and netropsin (Nt) prefer binding to AT rich DNA segments, which are candidates for intrinsic DNA helix bends. But our data for Hoe interaction with calf thymus DNA (ctDNA) show characteristic differences to those for Nt-ctDNA interaction. Especially for Hoe, the mode of highest affinity is saturated already at a ligand concentration of roughly 1 nM (r = 0.0015 Hoe/DNA-P). It exhibits an unusually strong temperature dependence of the conformational DNA response. A Hoe-Nt competition experiment shows that Hoe binding to the sites of the very first Hoe mode is almost unaffected by bound Nt. But Hoe binding to the sites of the following Hoe modes does not occur due to the competition with Nt. Thus this mode of strongest Hoe-DNA interaction reflects a unique mechanism, possibly of high relevance for gene regulatory systems.     

Structural transitions of chromatin induced by netropsin

Structural changes of chromatin induced by interaction of netropsin (Nt) with DNA has been examined by analysis of CD and electromicroscopic measurements. The results demonstrate the existence of a transition from the condensed globular state of chromatin into nucleosomal fibres generated by extremely low Nt concentration up to 1 mole Nt per 200 nucleotides. A second transition occurs at high Nt ratio per DNA phosphate (v' = 0.3). involving disorganization of nucleosomal particles. The interference of the Nt binding with chromatin proteins maintaining the sub- and superstructure will be discussed.     

Structure of the complexes of distamycin type antibiotics and actinomycin D with DNA: new data on the localization of these antibiotics within the DNA narrow groove]

It is shown that antibiotics actinomycin D (AM), netropsin (Nt), distamycin A (DM) and the propyl analogue of distamycin A (pDM) being complexed with DNA are located within the narrow groove of DNA. A comparative investigation of the 3H-dimethyl sulphate methylation extent of free calf thymus DNA and its complexes with AM, Nt, DM and pDM reveals that upon DNA saturation these antibiotics decrease the methylation level of the narrow groove (AM by 30%, pDM by 50%, DM by 65% and Nt by 70%). In the triple complex of DNA+AM+DM the methylation level of the narrow groove drops by 80%. The large groove is not shielded by these antibiotics at all. However, the methylation level of the large groove decreases by 50% for T6 phage DNA due to the presence of glucosyl residues linked to 5-hydroxymethylcytosine within the large groove. The binding of AM to DNA saturated with Nt or with the analogue of distamycin A (DM2) containing the 2 N-methylpyrrole residues has been investigated by spectrophotometry. The apparent number of binding sites for AM in these 2 complexes is about half as much as observed for free DNA while the saturation level of the binding decreased only by about 20%. This proves simultaneous presence of AM and Nt (DM2) within the narrow groove of DNA.     

Experimental studies on the nature of bonding of DNA*bipyridyl-(ethylenediamine)platinum(II) and DNA*netropsin complexes in solution and oriented wet-spun films

The stability of complexes of NaDNA with bipyridyl- (ethylenediamine)platinum(II) (abbreviated [(bipy)Pt(en)](2+)) and with netropsin has been studied using two techniques: (i) ultraviolet (UV) melting experiments were done on NaDNA* [(bipy)Pt(en)](2+), showing that the [(bipy)Pt(en)](2+) ligand stabilizes the DNA double helix structure; and (ii) swelling measurements (via optical microscopy) as a function of relative humidity were done on wet-spun oriented films of NaDNA*[(bipy)Pt(en)](2+) and of NaDNA*netropsin. The swelling data shows that an irreversible transition of the films occurs at high relative humidity, first for the NaDNA*netropsin, then for pure NaDNA, and lastly for the NaDNA*[(bipy)Pt(en)](2+). These results are indicative that the [(bipy)Pt(en)](2+) complex stabilizes the intermolecular bonds which mediate the film swelling characteristics. A model is suggested for the binding of [(bipy)Pt(en)](2+) to DNA to explain why the swelling experiments show this ligand as increasing the intermolecular bond strength between the DNA double helices, while netropsin decreases this degree of stabilization.     

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.     

Z-DNA and other non-B-DNA structures are reversed to B-DNA by interaction with netropsin

The interaction between the B-form specific ligands netropsin (Nt) and distamycin-3 (Dst-3) and DNA duplexes has been studied under conditions of salt concentration and low water activity that modify the polymer conformation into a non-B DNA form, putatively a Z-like form. Three polymers with strict alternating purine-pyrimidine sequences and GC content from 100-0% have been tested: poly(dG-dC) . poly(dG-dC), poly(dA-dC) . poly(dG-dT) and poly(dA-dT) . poly(dA-dT). The titrations by Nt and Dst-3 were followed by circular dichroism. Although specific binding of Nt to the Z-form of poly(dG-dC) . poly(dG-dC) does not occur, Nt reverses this Z structure to the B-type conformation; Dst-3 is, however, totally inefficient. The presumed non-B or Z-like structure of poly(dA-dC) . poly(dG-dT) is reversed to the B-form upon interaction with Nt; Dst-3 also induces this reversal but at higher ligand ratios. The modified B-structure of poly(dA-dT) . poly(dA-dT) in low water activity is efficiently reversed to the B-form by interaction with both Nt and Dst-3.     

Secondary structure of DNA from T4 and T6 phages]

The secondary structure of NaDNA from E. coli T4 and T6 phages has been studied by the X-ray diffraction method. Molecules of these DNAs as well as T2 phage DNA molecules contain hydroxymethylcytosine glucosylated at different position instead of cytosine. At high relative humidity these DNAs are shown to exist in B-conformaion. As humidity decreases the transformation into T=conformation takes place in the T4 phage DNA whereas in the T6 phage DNA changes of secondary structure similar to B-T transformation occur which do not result however in the appearance of all the characteristics of the T-conformation.     

Alteration of A.T base-pair opening kinetics by the ammonium cation in DNA A-tracts

We have investigated by NMR the effects of NH(4)(+) on the chemical shifts, on the structure, and on the imino proton exchange kinetics of two duplexes containing an A-tract, [d(CGCGAATTCGCG)](2) and [d(GCA(4)T(4)GC)](2), and of a B-DNA duplex,[d(CGCGATCGCG)](2). Upon NH(4)(+) addition to [d(CGCGAATTCGCG)](2), the adenosine H2 protons, the thymidine imino protons, and the guanosine imino proton of the adjacent G.C pair show unambiguous chemical shifts. Similar shifts are observed in the A-tract of [d(GCA(4)T(4)GC)](2) and for the A5(H2) proton of the B DNA duplex [d(CGCGATCGCG)](2). The localization of the shifted protons suggests an effect related to NH(4)(+) binding in the minor groove. The cross-peak intensities of the NOESY spectra collected at low and high NH(4)(+) concentrations are comparable, and the COSY spectra do not show any change of the sugar pucker. This indicates a modest effect of ammonium binding on the duplex structures. Nevertheless, the imino proton exchange catalysis by ammonia provides evidence for a substantial effect of NH(4)(+) binding on the A.T base-pair kinetics in the A-tracts. Proton exchange experiments performed at high and low NH(4)(+) concentrations show the occurrence of two native conformations in proportions depending on the NH(4)(+) concentration. The base-pair lifetimes and the open-state lifetimes of each conformation are distinct. Exchange from each conformation proceeds via a single open state. But if, and only if, the NH(4)(+) concentration is kept larger than 1 M, the A.T imino proton exchange times of A-tract sequences exhibit a linear dependence versus the inverse of the NH(3) proton acceptor concentration. This had been interpreted as an indication for two distinct base-pair opening modes (W?rml?nder, S., Sen, A., and Leijon, M. (2000) Biochemistry 39, 607-615).     

A 23Na NMR study of the effect of D(+) and L(-) arabitol on NaDNA in aqueous solution.

The 23Na NMR quadrupolar relaxation in NaDNA aqueous solutions has been investigated in the presence of D(+) and L(-) arabitol. Quite different results were produced by the enantiomers, i.e. the addition of D(+) arabitol produced a small increase of the 23Na NMR relaxation rates, while in the presence of L(-) arabitol a significant decrease was observed. These findings were analysed and discussed in terms of an effective interaction of L(-) arabitol with DNA.     

Optical third harmonic generation study of the hydration of DNA films.

Optical third harmonic generation (THG) has been observed for the first time from DNA films. The THG signal is observed from NaDNA films exposed to relative humidities (RHs) between 0% and 98%. A strong enhancement (approximately 5x) of the THG signal from NaDNA is observed at 84% RH; no enhancement is observed for RbDNA. The most likely mechanism for such an enhancement is an increased coherence length. A model calculation using estimates of the refractive indices at both the fundamental and third harmonic frequencies supports this interpretation. The observed THG signal has the same polarization as the incident (fundamental) light. For the A conformation, the THG signal polarized perpendicular to the helical axis is approximately twice as strong as the signal polarized parallel to the helical axis. No such anisotropy is observed for either the disordered conformation (below about 50% RH) or the B conformation (above 92% RH).     

Aspects of specific protein-DNA interaction; multi-mode binding of the oligopeptide antibiotic netropsin to (A.T)-rich DNA segments.

By means of titration viscometry a number of distinct modes could be resolved for the interaction between the antibiotic netropsin and DNA species of 50, 58, and 69 mole + (A+T) below r = 0.04 netropsin molecules bound per DNA phosphate group. The number of corresponding binding sites increases with a high power of the (A+T) content. The apparent association constants are very high (greater than 10(6) M-1, some perhaps greater than 10(6) M-1) and also rather different for most of the binding sites. It is suggested that some of these interaction modes differ in the number of hydrogen bonds formed between donors of the ligand and acceptors of the binding sites. The interaction modes were characterized quantitatively by their (species-independent) changes of DNA contour length and by the percentage of local DNA stiffening.     

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´′- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´′-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.     

The effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I

Nt.BspD6I nicking endonuclease stimulates template/primer-independent DNA synthesis by Bst DNA polymerase. Template/primer-independent DNA synthesis may be one of the reasons for the formation of nonspecific products in certain DNA amplification reactions, especially those involving nicking endonucleases. Expansion of the range of DNA amplification procedures performed in the presence of nicking endonucleases makes the search for template/primer-independent DNA synthesis inhibitors highly relevant. The present work has shown that a single-strand DNA binding protein from E. coli does not affect template/primer-independent DNA synthesis regardless of the presence or absence of Nt.BspD6I. A single-stranded DNA-binding protein coded by gene 32 from bacteriophage T4 completely inhibits template/primer-independent DNA synthesis in the absence of nicking endonuclease. If nicking endonuclease is present, the protein does not suppress the synthesis of the specific product but causes a significant decrease of the amount of template/primer-independent DNA synthesis products.     

Influence of nucleotide sequence on dA.dT-specific binding of Netropsin to double stranded DNA.

Using CD measurements the complex formation of Netropsin (Nt) with poly(dA-dC).poly(dT-dG) and its stability against high salt concentrations is compared with that of poly(dA).poly(dT) and poly(dA-dT).POLY(DT-dA). It is experimentally shown that the insertion of a dG.dC pair in dA.dT sequences strongly reduces the specific interaction of Nt with DNA duplexes. The specificity of the interaction is strongly increased by two or more consecutive thymine residues as present in thymine isostichs of double stranded DNA's.     

Effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I

In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.     

Competitive interactions of Co(NH3)6(3+) and Na+ with helical B-DNA probed by 59Co and 23Na NMR

59Co NMR is demonstrated to provide a useful probe of the interactions of Co(NH3)6(3+) with helical B-DNA. The association of Co(NH3)6(3+) with B-DNA produces relatively modest effects on the relaxation rate and chemical shift of 59Co, which indicate that the octahedral coordination shell remains intact and that no significant number of long-lived "outer-sphere" complexes are formed at specific sites on the DNA surface. Under conditions where essentially all of the cobalt complex is associated with DNA, the chemical shift of 59Co appears to depend on its binding density. This effect could be due to magnetic heterogeneity in the environments of Co(NH3)6(3+) adjacent to DNA. The local exchange reaction between Co(NH3)6(3+) and Na+ in the vicinity of DNA has been investigated by measuring 59Co chemical shifts and 23Na line widths concurrently. The number of sodium ions displaced by the association of one Co(NH3)6(3+) with DNA cannot be uniquely determined, but the data indicate that this number remains constant over at least the initial stage of a titration of NaDNA with NaCl. 59Co chemical shifts have been analyzed to construct binding isotherms for the association of cobalt hexaammine with DNA over a range of salt (NaCl) concentrations. The magnitudes of the resulting binding constants and their salt dependence are similar to those previously reported for the association of structurally diverse trivalent ligands, such as spermidine and trilysine, with helical nucleic acids. Therefore, these association equilibria appear to be governed primarily by electrostatic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)