Long-range allosteric effects on the B to Z equilibrium by daunomycin

Spectroscopic and fluorometric methods were used to study the binding of the anticancer drug daunomycin to poly[d(G-C)] and poly[d(G-m5C)] under a variety of solution conditions. Under high-salt conditions that favor the left-handed Z conformation, binding isotherms for the interaction of the drug with poly[d(G-C)] are sigmoidal, indicative of a cooperative binding process. Both the onset and extent of the cooperative binding are strongly dependent upon the ionic strength. The binding data may be explained by a model in which the drug preferentially binds to B-form DNA and acts as an allosteric effector on the B to Z equilibrium. At 2.4 M NaCl, binding of as little as one drug molecule per 20 base pairs (bp) results in the conversion of poly[d(G-C)] from the Z form entirely to the B form, as inferred from binding data and demonstrated directly by circular dichroism measurements. Similar results are obtained for poly[d(G-m5C)] in 50 mM NaCl and 1.25 mM MgCl2. Under these solution conditions, it is possible to demonstrate the Z to B structural transition in poly[d(G-m5C)] as a function of bound drug by the additional methods of sedimentation velocity and susceptibility to DNase I digestion. The transmission of allosteric effects over 20 bp is well beyond the range of the drug's binding site of 3 bp. Since daunomycin preferentially binds to alternating purine-pyrimidine sequences, which are the only sequences capable of the B to Z transition, the allosteric effects described here may be of importance toward understanding the mechanism by which the drug inhibits DNA replicative events.(ABSTRACT TRUNCATED AT 250 WORDS)     

The B-Z conformational transition and aggregation of poly[d(G-C)] induced by moderate concentrations of Mg(CIO4)2

Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions.     

Allosteric conversion of Z DNA to an intercalated right-handed conformation by daunomycin

Absorbance and fluorescence methods were used to measure the binding of the anticancer drug daunomycin to poly (dGdC) under ionic conditions that initially favor the left-handed Z conformation of the polymer. Drug binding was cooperative under these conditions and may be fully accounted for by an allosteric model in which the drug binds preferentially (but not exclusively) to the right-handed B conformation and shifts the polymer from the Z to an intercalated right-handed conformation. Quantitative analysis of binding isotherms in terms of the allosteric model allowed for estimation of the equilibrium constants for the conversion of a base pair at a B-Z interface from the Z to the B conformation and for the formation of a base pair in the B conformation within a stretch of helix in the Z conformation. The free energy of the Z to B conversion of a base pair was calculated from this data and ranges from +0.03 to +0.3 kcal/mol over the NaCl range of 2.4-3.5 M. The free energy for the formation of a B-Z junction was nearly constant at +4.0 kcal/mol over the same range of NaCl concentrations. The salt dependence of the free energy of the Z to B transition indicates preferential Na+ binding to the Z form and that there is a net release of Na+ upon conversion of a base pair from the Z to the B conformation. The energetically unfavorable Z to B transition was found by this analysis to be driven by coupling to the energetically favorable interaction of daunomycin with B form DNA. In 3.5 M NaCl, for example, the free energy change for the overall reaction (Z DNA base pairs) + (daunomycin) in equilibrium with (right-handed complex) is -7.0 kcal/mol, nearly all of which is contributed by the binding of drug to B DNA. Analysis using the allosteric model also shows that the number of base pairs converted from the Z to the B conformation per bound drug molecule is salt dependent and provides evidence that drug molecules partition into regions of the polymer in the right-handed conformation.     

Conformational lability of poly(dG-m5dC):poly(dG-m5dC).

The remarkable conformational lability of poly(dG-m5dC):poly(dG-m5dC) is demonstrated by the observation of an acid-mediated conformational hysteresis. An acid-mediated Z conformation that exists in solutions containing low sodium concentrations that would normally favor the B conformation is described in this report. This Z conformation is reached by an acid-base titration of a B-poly(dG-m5dC):poly(dG-m5dC) solution which is not far from the B-Z transition midpoint. The resulting Z conformation is thermally very stable, with direct melting into single strands at approximately 100 degrees C. In contrast, the B form DNA, initially in solutions of the same ionic strength but without exposure to acidic pH, exhibits a biphasic melting profile, with conversion into the Z form (with high cooperativity) prior to an eventual denaturation into single strands at around 100 degrees C. Cooling experiments reveal that such biphasic transitions are quite reversible. The transition midpoint for the thermally poised B to Z transformation depends strongly on the NaCl concentration and varies with sample batch. The acid-mediated Z form binds ethidium more weakly than its B counterpart, and the ethidium induced Z to B conversion occurs in a step-wise (non-allosteric) fashion without the requirement of a threshold concentration. The acid-mediated as well as the thermally poised Z conformations are reversed by the addition of EDTA, suggesting the involvement of trace amounts of multivalent metal ions.     

Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.     

Mn2+ and other transition metals at low concentration induce the right-to-left helical transformation of poly[d(G-C)].

The effects of the first-row transition metal ions on the right(B)- to left(Z)-handed helical transition of poly[d(G-C)] have been determined. The Z conformation is induced by MnCl2 at submillimolar concentrations. The forward reaction has a very large activation energy (440 kJ/mol) so that a facile conversion occurs only at temperatures above 45 degrees C. However, the left-handed form remains stable upon cooling. The addition of ethanol (20% v/v) eliminates the requirement for elevated temperature. The transition is highly co-operative and is accompanied by spectral changes (absorption, circular dichroism) characteristic for the B----Z conformational transition. NiCl2 and CoCl2 also induce the B----Z transition in poly[d(G-C)] but the activation energies and thus the temperature requirements for the forward reaction are lower than those observed with MnCl2. The left-handed DNA formed in the presence of Mn2+ is similar to 'Z DNA' previously described in Mg2+-EtOH (van de Sande and Jovin , 1982): (a) it readily sediments out of solution at low speed as a consequence of intermolecular association which, however, is not accompanied by turbidity; and (b) it supports the binding of ethidium bromide although this drug interacts preferentially with the B form of DNA. With Ni2+, the B----Z isomerization step can be separated from the subsequent specific Z----Z* association. Mn2+, Ni2+, and Co2+ also promote the B----Z transition of poly[d(G-m5C)] at substoichiometric concentrations with respect to DNA nucleotide.     

500 MHz 1H-NMR study of the interaction of daunomycin with B and Z helices of d(CGm5CGCG)

The interaction of daunomycin with B and Z helices of a self-complementary DNA fragment d(CGm5CGCG) in solution was studied by 1H-NMR spectroscopy at 500 MHz. The results show that the B-Z transition kinetics is not affected by addition of daunomycin. Daunomycin binds exclusively to the B form of d(CGm5CGCG). Z exchanges with B while the latter also exchanges with the B duplex-daunomycin complexes.     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Chromomycin A3 binds to left-handed poly(dG-m5dC)

The interaction of chromomycin A3 (an antitumor antibiotic) with right-handed and left-handed polynucleotides has been studied by absorbance, fluorescence, circular dichroism, 31P-NMR and 1H-NMR techniques. Binding to either the B form of poly(dG-dC) or the Z form of poly(dG-m5dC) shifts the absorbance maximum to higher wavelength and enhances the fluorescence of the drug. Circular dichroic spectra of solutions containing various concentrations of chromomycin A3 and fixed concentrations of either B or Z polynucleotides show well defined isoelliptic points at similar wavelengths. At the isoelliptic point, the drug complex with B DNA exhibits positive ellipticity while with Z DNA it exhibits negative ellipticity. 31P-NMR spectra of the chromomycin A3 complex with the Z form of poly(dG-m5dC) demonstrate that the Z conformation is retained in the drug complex up to one molecule drug/four base pairs. At Mg2+ concentrations lower than that necessary to stabilize the left-handed conformation of poly(dG-m5dC) alone, 31P analysis shows that chromomycin A3 can bind simultaneously to both the B and Z conformations of poly(dG-m5dC), with no effect on the B-Z equilibrium. These data demonstrate that chromomycin A3 binds to left-handed poly(dG-m5dC) with retention of the left-handed conformation up to saturating drug concentrations.     

A left-handed (Z) conformation of poly(dA-dC).poly(dG-dT) induced by polyamines.

Blocks of potential Z-DNA forming alternating purine-pyrimidine (APP) sequences are widely dispersed in native DNAs. We have studied the effects of naturally occurring polyamines on the conformation of a synthetic APP sequence, poly(dA-dC).poly(dG-dT) by circular dichroism spectroscopy. In the presence of micromolar concentrations of spermidine (125 microM) and spermine (16 microM), this polymer undergoes B to Z transition in low ionic strength (2 mM Na+) buffers. The concentration of polyamines required for B to Z transition increases with Na+ in the buffer and a straight line is obtained on plotting ln[Na+] vs. ln [spermidine 3+]. However, at concentrations of polyamines higher than those necessary to induce B to Z transition, Z-DNA converts to psi-DNA, an ordered, twisted, tight packing arrangement of the double helix. These results suggest a pathway for the transient formation of Z-DNA segments in vivo by interaction of the ubiquitous polyamines with naturally occurring blocks of APP sequences.     

A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine-induced B-DNA to Z-DNA conformational transition of a plasmid DNA with (dG-dC)n insert.

We investigated the ability of natural polyamines putrescine, spermidine, and spermine to provoke a left-handed Z-DNA conformation in a recombinant plasmid (pDHg16) with a 23-base pair insert of (dG-dC)n.(dG-dC)n sequences. Using a monoclonal anti-Z-DNA antibody (Z22) and an enzyme-linked immunosorbent assay protocol, we found that spermidine and spermine were capable of converting pDHg16 to the Z-DNA form. The concentrations of spermidine and spermine at the midpoint of the B-DNA to Z-DNA transition were 280 and 5 microM, respectively, in buffer containing 50 mM NaCl, 1 mM sodium cacodylate, and 0.15 mM EDTA, pH 7.4. A plot of ln[Na+] versus ln [spermine4+], where [Na+] is the bulk NaCl concentration and [spermine4+] is the spermine concentration at the midpoint of the B-DNA to Z-DNA transition, gave a straight line with a slope of 1.2. Structural specificity was clearly evident in the efficacy of three spermidine homologs to induce the Z-DNA conformation in pDHg16. Putrescine and acetylspermidines had no effect on the conformation of the plasmid DNA up to a 3 mM concentration. Control experiments with the parental plasmid (pDPL6) showed no binding of the plasmid DNA with Z22. These results indicate that spermidine and spermine are capable of provoking the left-handed Z-DNA conformation in small blocks of (dG-dC)n sequences embedded in a right-handed B-DNA matrix. Since blocks of (dG-dC)n sequences are found in certain native DNAs, conformational alterations of these regions to the Z-DNA form in the presence of polyamines may have important gene regulatory effects.     

Z* DNA, the left-handed helical form of poly[d(G-C)] in MgCl2-ethanol, is biologically active.

The interconversion between the right (R) and left (L) helical forms of poly[d(G-C)] occurs at low concentrations of MgCl2 and EtOH, acting together in a highly synergistic manner. Thus, the cooperative R---L transition is induced by only 0.4 mM and 4 MM MgCl2 in combination with 20% and 10% EtOH, respectively. The L form of poly[d(G-C)] formed under these conditions has the spectroscopic properties (absorption, circular dichroism) previously demonstrated under high salt conditions (Pohl and Jovin, 1972) and thought to correspond to the left-handed Z DNA structures recently established by X-ray crystallography (Wang et al., 1979; Drew et al., 1980). However, L DNA formed in Mg2+-EtOH (which we designate as Z* DNA) has unique properties: a) it can be sedimented readily out of solution at low speed, indicative of condensation and intermolecular aggregation; b) it supports the binding of several intercalating (ethidium bromide, actinomycin D) and non-intercalating (mithramycin) drugs, although these interact preferentially with the R (i.e., B) form of DNA; and c) it functions as a template for Escherichia coli RNA polymerase. B and Z* DNAs can be generated under identical ionic conditions and compared in a number of biochemical systems. Our results suggest that left-handed DNA may form under physiological conditions and serve a biological function.     

Multivalent ions are necessary for poly[d(AC).d(GT)] to assume the Z form: a CD study.

In previous work, it was shown that poly [d(AC) · d(GT)] could be forced into the Z form by strong dehydrating conditions, provided EDTA was not present. Presumably multivalent impurities were also necessary for the transition. In order to gain control over the B to Z transition for this DNA, we carefully removed all divalent contaminants from the sample and asked the obvious question: What ions are necessary for the transition under dehydrating conditions? We systematically investigated the effect of various multivalent ions. The common contaminants Ca²⁺, Mg²⁺, and Fe³⁺ will not cause the transition, but Co²⁺ and Ni²⁺ facilitate the transition, undoubtedly because of their well-known propensity to bind to purine N7. Since the transition also depends on the synergistic dehydrating action of sodium perchlorate and ethanol, we include CD spectra for the independent variations of these two factors. In addition, vacuum-uv CD spectra for the A form and various B forms of poly [d (AC) · d (GT)] are presented for the first time.     

Flexibility of DNA and RNA upon binding to different metal cations. An investigation of the B to A to Z conformational transition by Fourier transform infrared spectroscopy

The interaction of DNA and RNA with Cu(II), Mg(II), [Co(NH3)6]3+ [Co(NH3)5Cl]2+ chlorides and, cis- and trans-Pt(NH3)2Cl2 (CIS-DDP, trans-DDP) has been studied by Fourier Transform Infrared (FT-IR) spectroscopy and a correlation between metal-base binding and conformational transitions in the sugar pucker has been established. It has been found that RNA did not change from A-form on complexation with metals, whereas DNA exhibited a B to Z transition. The marker bands for the A-form (C3'-endo-anti conformation) were found to be near 810-816 cm-1, while the bands at 825 and 690 cm-1 are marker bands for the B-conformation (C2'-endo, anti). The B to Z (C3'-endo. syn conformation) transition is characterized by the shift of the band at 825 cm-1 to 810-816 cm-1 and the shift of the guanine band at 690 cm-1 to about 600-624 cm-1.     

Interactions of chromomycin A3 and mithramycin with the sequence d(TAGCTAGCTA)2

Anti-cancer antibiotics, chromomycin A3 (CHR) and mithramycin (MTR) inhibit DNA directed RNA synthesis in vivo by binding reversibly to template DNA in the minor groove with GC base specificity, in the presence of divalent cations like Mg2+. Under physiological conditions, (drug)2Mg2+ complexes formed by the antibiotics are the potential DNA binding ligands. Structures of CHR and MTR differ in their saccharide residues. Scrutiny of the DNA binding properties reveal significant differences in their sequence selectivity, orientation and stoichiometry of binding. Here, we have analyzed binding and thermodynamic parameters for the interaction of the antibiotics with a model oligonucleotide sequence, d(TAGCTAGCTA)2 to understand the role of sugars. The oligomer contains two potential binding sites (GpC) for the ligands. The study illustrates that the drugs bind differently to the sequence. (MTR)2Mg2+ binds to both sites whereas (CHR)2Mg2+ binds to a single site. UV melting profiles for the decanucleotide saturated with the ligands show that MTR bound oligomer is highly stabilized and melts symmetrically. In contrast, with CHR, loss of symmetry in the oligomer following its association with a single (CHR)2Mg2+ complex molecule leads to a biphasic melting curve. Results have been interpreted in the light of saccharide dependent differences in ligand flexibility between the two antibiotics.     

A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation.

The nature of binding of Ru(phen) 2+ (I), Ru(bipy) 2+ (II), Ru(terpy) 2+ (III) (phen = 1,10-phenanthroline, bipy 3 = 2,2'-bipyridyl, 3 terpy = 2,2'2," - 2 terpyridyl) to DNA, poly[d(G-C)] and poly[d(A-T)] has been compared by absorption, fluorescence, DNA melting and DNA unwinding techniques. I binds intercalatively to DNA in low ionic strength solutions. Topoisomerisation shows that it unwinds DNA by 22 degrees +/- 1 per residue and that it thermally stabilizes poly[d(A-T)] in a manner closely resembling ethidium. Poly[d(A-T)] induces greater spectral changes on I than poly[d(G-C)] and a preference for A-T rich regions is indicated. I binding is very sensitive to Mg2+ concentration. In contrast to I the binding of II and III appears to be mainly electrostatic in nature, and causes no unwinding. There is no evidence for the binding of the neutral Ru(phen)2 (CN)2 or Ru(bipy)2 (CN)2 complexes. DNA is cleaved, upon visible irradiation of aerated solutions, in the presence of either I or II.