Christoph Zimmer

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.

DNA binding properties of minor groove binders and their influence on the topoisomerase II cleavage reaction.

We present titrations of the human δβ‐globin gene region with DNA minor groove binders netropsin, bisnetropsin, distamycin, chromomycin and four bis‐quaternary ammonium compounds in the presence of calf thymus topoisomerase II and DNase I. With increasing ligand concentration, stimulation and inhibition of enzyme activity were detected and quantitatively evaluated. Additionally we show a second type of stimulation, the appearance of strong new topoisomerase II cleavage sites at high ligand concentrations. The specific binding sites of the minor groove binders of the DNA sequence and their microscopic binding constants were determined from DNase I footprints. A binding mechanism for minor groove binders is proposed in order to explain these results especially when ligand concentration is increased.

Parallel‐stranded duplex DNA containing dA · dU base pairs

DNA oligonucleotides with dA and dU residues can form duplexes with trans d(A.U) base pairing and the sugar-phosphate backbone in a parallel-stranded orientation, as previously established for oligonucleotides with d(A.T) base pairs. The properties of such parallel-stranded DNA (ps-DNA) 25-mer duplexes have been characterized by absorption (uv), CD, ir, and fluorescence spectroscopy, as well as by nuclease sensitivity. Comparisons were made with duplex molecules containing (a) dT in both strands, (b) dU in one strand and dT in the second, and (c) the same base combinations in reference antiparallel-stranded (aps) structures. Thermodynamic analysis revealed that total replacement of deoxythymine by deoxyuridine was accompanied by destabilization of the ps-helix (reduction in Tm by -13 degrees C in 2 mM MgCl2, 10 mM Na-cacodylate). The U-containing ps-helix (U1.U2) also melted 14 degrees C lower than the corresponding aps-helix under the same ionic conditions; this difference was very close to that observed between ps and aps duplexes with d(A.T) base pairs. Force field minimized structures of the various ps and aps duplexes with either d(A.T) or d(A.U) base pairs ps/aps and dT/dU combinations are presented. The energy-minimized helical parameters did not differ significantly between the DNAs containing dT and dU.

Interaction of nucleic acids with a non-intercalative anti-leukemic compound containing bisquarternary heterocycles

The binding of an antitumour drug with bisquarternary ammonium heterocyclic structure, NSC-101327, to nucleic acids has been examined by using ultraviolet absorption and CD measurements. Like the minor groove-binding oligopeptides, netropsin and distamycin A, the optically inactive chromophoric system of NSC-101327 shows induced Cotton effects in the CD spectra of complexes with various DNAs, RNA and single-stranded polynucleotides. This property directly reflects interaction of NSC-101327 with different types of nucleic acids at moderate ionic strength, which contrasts with previous findings of a higher selective binding of netropsin to B-DNA. However, an efficient interactin of NSC-101327 with dA X dT basepair sequences is demonstrated by a large melting temperature increase of dA X dT-rich DNAs. NSC-101327 also reacts with dG X dC base pairs of B-DNA and forms a complex with Z-DNA of poly( br8dG -dC) X poly( br8DG -dC). The affinity of NSC-101327 to poly(dG-dC) X poly(dG-dC) is, however, lower, and the CD spectral binding effect depends on the ionic strength. The CD results of the complex with poly(dA-dT) X poly(dA-dT) suggests at least two binding modes, in accordance with previous conclusions. This is indicated by a clear-cut initial increase of the CD signal and a subsequent large decrease to negative CD signals. Competition experiments with netropsin suggest that binding of NSC-101327 occurs preferentially in the minor groove without intercalation. NSC-101327 also tends to interact with lower binding affinity to dG-dC pairs in B-DNA, with rA X rU pairs of RNA and with single-stranded polynucleotides. Thus our results suggest that NSC-101327 represents a DNA groove-binding ligand of lower basepair specificity and lower conformational selectivity compared to the B-specific netropsin probe.

Magnetic circular dichroism study of the binding of netropsin and distamycin-A with DNA

The magnetic circular dichroism (MCD) of netropsin and distamycin-A is reported. New data for the interaction with dA ; dT base pairs in DNA were obtained from the MCD of their complexes with DNA duplex polymers. The MCD results allow an interpretation of the induced Cotton effects in the natural CD spectra of netropsin and distamycin-A complexes with DNA. While large distortions of the bases in DNA by the oligopeptide interaction is excluded, some subtle conformational variations of the DNA might explain the inhibition of the enzyme function of netropsin and distamycin-A on DNA.

Interaction of phenosafranine with nucleic acids and model polyphosphates: III. Heterogeneity in phenosafranine interactions with DNA base pairs

Fluorescence and circular dichroism spectral measurements, thermal denaturation studies and binding competition experiments with netropsin and actinomycin D were carried out in systems containing phenosafranine bound to DNAs differing in base composition. The investigated properties exhibit a heterogeneity related to the content of A.T and G.C pairs in DNA and to the nature of phenosafranine binding modes. At low level of saturation of binding sites (r less than 0.1) phenosafranine does not show strong preference for any of the DNA base pairs in the overall binding. However, the strong monomer non-cooperative binding outside the helix (mode I1) occurs predominantly, even though not exclusively in G.C rich regions. The strong binding modes involving intercalated dye molecules (mode I2 and eventually mode II1) prevail in A.T rich regions. These binding modes become the principal types of strong phenosafranine interaction with DNA when the level of saturation of binding sites increases, i.e. at r greater than 0.1.20

Different Effects of Nonintercalative Antitumor Drugs on DNA Triple Helix Stability: SN-18071 Promotes Triple Helix Formation

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN-18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA).2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA).poly(dT) in a different manner. SN-18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic interactions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.

Protection of (dA.dT) cluster regions in the DNAase I cleavage of DNA by specific interaction with netropsin.

The specific DNA binding ligand netropsin selectively blocks dA-dT base pairs in clusters containing two or more consecutive thymine residues at the dNAase I cleavage sites of DNA. Using CD and UV absorption measurements it is shown, that at various ratios of netropsin to nucleotide concentrations and even at satuation of ligand interaction the enzyme cuts along regions containing dG-dC pairs sandwiched between dA-dT pairs. This follows a slow kinetics and is associated with a release of netropsin from those segments. These facts suggests the usefulness of the partial protection of certain DNA sequences in DNAase I cleavage sites in producing DNA fragments in structural studies of the genome. A possible interpretation of the effect of netropsin binding on the enzymatic hydrolysis of phosphodiester bonds of the helix is discussed.

Reversal of the Z- to B-Conformation of Poly(dA-dT)•Poly(dA-dT) Induced by Netropsin and Distamycin A

Abstract Poly(dA-dT)•poly(dA-dT) which adopts the Z-form at 5 M NaCl in presence of 95 mM Ni2+ions is reversed to the B-conformation by the nonintercalating drugs netropsin (Nt) and distamycin A (Dst). The drug-induced reversal from the Z-to B-form of poly(dA- dT)•poly(dA-dT) is evidenced by CD spectral changes at characteristic wavelengths around 295 nm and 248 nm. The drug-induced conformational transition is accompanied by a slow kinetic process. The results suggest the preference of these AT-specific drugs for the B-form and the inability of Nt and Dst to form a stable complex with the Z-form of poly(dA-dT)•poly(dA-dT).

DNA Binding of the Nonintercalative Ligands SN-6132, SN-6131 and SN-6113: Minor Variations of the Ligand Structure May Cause Changes in the Base Pair Preference

The DNA binding selectivity of three ligands of a series of antitumor agents of bisquaternary ammonium heterocycles has been investigated by means of CD spectroscopy and melting measurements. From the spectroscopic results and binding data it is concluded that the agents SN-6132, SN-6131 and SN-6113 have relatively high affinity to AT base pair sequences whereas the binding to GC pairs is very low. The binding selectivity to AT base pair sequences decreases in the order netropsin > SN-6132 > SN-6113 > SN-6131. Poly(dA).poly(dT) has the highest binding preference for SN-6132 relative to that of SN-6131. The different binding behavior of the ligands is related to their distinct changes in the chemical structure and to the DNA minor groove properties which determines the adaptability of the ligands in the groove.