Interaction of DNA methyltransferase with aminofluorene and N-acetylaminofluorene modified poly(dC-dG)

Poly(dC-dG) was reacted in vitro to yield templates containing similar amounts of aminofluorene (AF) or acetylaminofluorene (AAF) adducts. These modified poly(dC-dG) templates were tested in an in vitro DNA methylation system utilizing 1500-fold purified rat liver methyltransferase (DMase) to compare and quantitate the effects of these adducts on the kinetics of methylation and the interaction of DMase with such templates. Enzymatic methylation is severely impaired by arylamine adducts, with bound AF inhibiting more than AAF (relative Vmax 0.24 for AAF-poly(dC-dG) and 0.066 for AF-poly(dC-dG). The apparent km for the reaction is not appreciably altered by AAF modification: 10 microM for dCdG dinucleotide units, but it is threefold lower (3 microM) for AF-poly(dC-dG). In competition experiments it was demonstrated that a translocational block is imposed by the adducts. From differential salt inhibition assays and preincubation assays, no change in the ionic binding to the altered templates could be detected, which suggests that the enzyme interacts very strongly through hydrophobic interactions with the fluorene ring. Evidence that the fluorene ring is exposed is supported by circular dichroism spectra of the templates under the conditions of the assay, which indicated that the AF adducts do not appreciably change the normal B conformation of the template, while the template with 9.5% modification by AAF adducts adopted a Z form. These results suggest that the inhibitory effects of AAF and, in particular, AF upon DMase-catalyzed methylation reactions are not dependent upon helix conformation. Instead, they appear to depend upon DMase recognition of an altered dG base configuration, which is responsible for altered binding and methylation kinetics.     

Conformation and dynamics of short DNA duplexes: (dC-dG)3 and (dC-dG)4

Natural abundance 13C NMR spectra of duplexed (dC-dG)3 and (dC-dG)4 exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(1) sugar. 4M NaCl causes changes in the 13C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the anti to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

Effects of 5 cytosine methylation on the B-Z transition in DNA restriction fragments and recombinant plasmids

Alternating (dC-dG)n regions in DNA restriction fragments and recombinant plasmids were methylated at the 5 position of the cytosine residues by the HhaI methylase. Methylation lowers the concentration of NaCl or MgCl2 necessary to cause the B-Z conformational transition in these sequences. Ionic strengths higher than physiological conditions are required to form the Z conformation when the methylated (dC-dG)n tract is contiguous with regions that do not form Z structures, in contrast to the results with the DNA polymer poly(m5dC-dG) . poly(m5dC-dG). In supercoiled plasmids containing (dC-dG)n sequences, methylation reduces the number of negative supercoils necessary to stabilize the Z conformation. Calculations of the observed free energy contributions of the B-Z junction and cytosine methylation suggest that two junctions offset the favorable effect of methylation on the Z conformation in (dC-dG)n sequences (about 29 base-pairs in length). Studies with individual methylated topoisomers demonstrate that increasing Na+ concentration up to approximately 0.2 M inhibits the formation of the Z conformation in the (m5dC-dG)n region of supercoiled plasmids. The results suggest that methylation may serve as a triggering mechanism for Z DNA formation in supercoiled DNAs.     

Z-DNA is formed by poly (dC-dG) and poly (dm5C-dG) at micro or nanomolar concentrations of some zinc(II) and copper(II) complexes

We report studies on the interaction of some zinc(II) and copper(II) complexes of amines and amino acids with poly(dC-dG) and poly(dm5C-dG). Of the zinc complexes the species zinc-tris(2-aminoethyl) amine is found to be the most efficient for inducing Z-DNA giving a mid point at low ionic strength of 1.4 microM (poly(dC-dG] and 44nM (poly(dm5C-dG). While an antagonistic effect on raising the ionic strength is observed, the transition occurs at only 2 microM for poly(dm5C-dG) at 150mM NaCl. The most efficient copper(II) complex is that of diethylene triamine, though copper(II) complexes are generally less efficient than zinc(II) complexes. We also report kinetic and thermodynamic studies upon the B-Z transition induced by these complexes. A model is proposed for the interaction of one of the zinc complexes which involves not only direct zinc-DNA binding but also the formation of hydrogen bonds between the metal bond amine groups and the residues adjacent to the coordination site.     

Z-DNA is Formed by poly(dC-dG) and poly (dm5C-dG) at Micro or Nanomolar Concentrations of some Zinc(II) and Copper(II) Complexes

Abstract

We report studies on the interaction of some zinc(II) and copper(II) complexes of amines and amino acids with poly(dC-dG) and poly(dm⁵C-dG). Of the zinc complexes the species zinc-tris(2-aminoethyl) amine is found to be the most efficient for inducing Z-DNA giving a mid point at low ionic strength of 1.4μM (poly(dC-dG)) and 44μM (poly(dm⁵C-dG). While an antagonistic effect on raising the ionic strength is observed, the transition occurs at only 2μM for poly(dm⁵C-dG) at 150mM NaCl. The most efficient copper(II) complex is that of diethylene triamine, though copper(II) complexes are generally less efficient than zinc(II) complexes. We also report kinetic and thermodynamic studies upon the B-Z transition induced by these complexes. A model is proposed for the interaction of one of the zinc complexes which involves not only direct zinc-DNA binding but also the formation of hydrogen bonds between the metal bond amine groups and the residues adjacent to the coordination site.     

Conformation and Dynamics of Short DNA Duplexes: (dC-dG)3 and (dC-dG)4

Abstract

Natural abundance ¹³C NMR spectra of duplexed (dC-dG)₃ and (dC-dG)₄ exhibit resolved resonances for most of the carbons at 0.1M NaCl in aqueous solution. Large transitions in chemical shift for many of the hexamer carbons (up to 1.8 ppm) are observed in variable temperature measurements. Determination of spin-lattice relaxation times and nuclear Overhauser enhancements in 0.1M NaCl indicate that the duplexes tumble almost isotropically, with overall correlation times near 5 nsec; the sugar carbons experience more rapid local motions than do the base carbons. The relaxation data are also consistent with the most rapid local motions occurring at the chain-terminal residues, especially in the Cyd(l) sugar. 4M NaCl causes changes in the ¹³C chemical shifts of most of the guanine base carbons, and rearrangements in the deoxyribose carbon shifts; this is consistent with changes predicted by a salt-induced B to Z transition, viz. conversion of the guanylates from the and to syn range about the glycosyl bond, and from the S to N pseudorotational state of the deoxyribose ring.     

31P-NMR analysis of the B to Z transition in double-stranded (dC-dG)3 and (dC-dG)4 in high salt solution

In 4M NaCl solutions (dC-dG)n (n = 3,4; approximately 9 mM) exist as a mixture o +/- B and Z forms. The low and high field components of two 31P NMR resonances originating from internal phosphodiester groups are assigned to the GpC and CpG linkages, respectively. Low temperatures stabilize the Z-forms, which completely disappear above 50 degrees C (n = 3) and 65 degrees C (n = 4). delta H = -44 and -17 kJ/mol for B to Z transition in the hexamer and octamer duplexes, respectively. Temperature dependent changes (0-50 degrees C range) in the spin-lattice relaxation times at 145.7 MHz are distinctly different for the 31P nuclei o +/- GpC and CpG groups. The relaxation data can be explained by assuming that the GpC phosphodiester groups undergo more local internal motion than do the CpG groups.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies

The effects of covalent modification of poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) by ethidium monoazide (a photoreactive analogue of ethidium) on the salt-induced B to Z transition are examined. Earlier studies have shown ethidium monoazide to bind DNA (in the absence of light) in a manner identical to that of the parent ethidium bromide. Photolysis of the ethidium monoazide-DNA complex with visible light results in the covalent attachment of the photoreactive analogue to the DNA. This ability to form a covalent adduct was utilized to probe the effects of an intercalating irreversibly bound adduct on the salt-induced B to Z transition of the poly(dGdC).poly(dGdC) and poly(dGm5dC).poly(dGm5dC) polynucleotides. In the absence of drug, the salt-induced transition from the B to Z structure occurs in a highly cooperative manner. In contrast, this cooperativity is diminished as the concentration of covalently attached drug is increased. The degree of inhibition of the B to Z transition is quantitated as a function of the concentration of covalently attached drug. At a concentration of one drug bound per four base pairs for poly(dGdC).poly(dGdC) and seven base pairs for poly(dGm5dC).poly(dGm5dC), total inhibition of this transition is achieved. Lower concentrations of bound drug were effective in the partial inhibition of this transition. The effects of the covalently bound intercalator on the energetics of the B to Z transition were determined and demonstrated that the adduct is effective in locking the alternating copolymer in a right-handed conformation under high salt conditions.     

The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases

N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.     

Structural forms and transitions for the complex of mercury(II) with poly(dG-dC)

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury(II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N3 or N7 for r less than or equal to 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D2O stabilized the Z structure for poly(dG-dC) complexed with HgCl2.     

The transitions between left- and right-handed forms of poly(dG-dC).

The circular dichroism study of water/trifluoroethanol (TFE) solutions of poly(dG-dC) has revealed the following: The polynucleotide is present as a B form up to a TFE content of 60% (v/v) or less. Then, a cooperative transition into a left-handed Z form occurs. Within the region of 66-78% TFE, a continuous non-cooperative change is going on which can be attributed to an intrafamily transition within the family of Z forms. At last, in the interval of 80-84% TFE, a second cooperative transition, probably, Z - A is realized. Both transitions, Z - A and Z - B, show slow kinetics (10-60 min) while the direct transitions from the A to B form taking less than 10 sec. The length of cooperativity for the B - Z transition, Vo = 25 base pairs was estimated using spermine molecules. Spermine was found to induce the B to Z transition in the (dG-dC) sequences even in the absence of TFE which might be biologically interesting.     

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.     

Energetic and structural inter-relationship between DNA supercoiling and the right- to left-handed Z helix transitions in recombinant plasmids

We have evaluated the B to Z conformational transitions in supercoiled recombinant plasmids containing different lengths of (dC-dG) described in the preceding paper. The sodium chloride-induced right- to left-handed transition in a small segment of the plasmids caused a relaxation of (-) supercoils which was monitored by electrophoretic mobility changes of individual topoisomers on agarose gels containing NaCl at concentrations up to 5.0 M. The number of supercoils relaxed was proportional to the length of the (dC-dG) segment in the plasmid in good agreement with theoretical values. A short B/Z junction region (less than 5 base pairs) was inferred. The stability of the Z conformation in (dC-dG) segments of the plasmids had a strong length dependency; shorter lengths were less stable. Ten base pairs of (dC-dG) was insufficient to allow a Z conformation under the conditions studied. Supercoiling imparts a substantial favorable free energy to the Z conformation, reducing the NaCl concentration necessary to cause the transition. The relationship of supercoiling with the NaCl concentration necessary to cause a B leads to Z transition suggests that supercoiling alone is sufficient to stabilize the Z conformation at physiological salt concentrations. These results support the notion that left-handed DNA has an important biological role.     

Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: kinetic studies

The photoaffinity analogue ethidium monoazide was used to prepare samples of poly(dGdC).poly(dGdC) containing covalently attached ethidium. The effects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly(dGdC).poly(dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC)4, (dGdC)5, and (dGdC)6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2 +/- 1.1 kcal mol-1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumably by locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.     

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.     

Conformational transitions of a synthetic DNA poly(dA-dU). poly(dA-dU) in concentrated solutions of caesium fluoride

Chiroptical properties of poly(dA-dU).poly(dA-dU) were studied in concentrated NaCl and CsF solutions to reveal the role of the alternating B conformation in the CsF-induced alternating B-X conformational transition of poly(dA-dT).poly(dA-dT). Poly(dA-dU).poly(dA-dU) has been chosen for this purpose because it has, instead of the alternating B conformation, a regular conformation like poly(dG-dC).poly(dG-dC) in low-salt solution. It was found that poly(dA-dU).poly(dA-dU) did not assume that Z form at high NaCl concentrations but exhibited extensive CsF-induced changes in the circular dichroism spectra like poly(dA-dT).poly(dA-dT). The changes of reflect two consecutive two-state conformational transitions of the polynucleotide, both taking place with fast kinetics and low cooperativity. The transition were interpreted as involving the regular and alternating B conformation at lower CsF concentrations and the alternating B and X conformation at higher CsF concentrations. A comparison of the behaviour of poly(dA-dU).poly(dA-dU) and poly(dA-dT).poly(dA-dT) in CsF solutions demonstrates that the thymine methyl groups promote the X form but are not crucial for its existence. On the other hand, the alternating B conformation appears to be the inevitable starting structure for DNA isomerization into the X form.     

Characterization of intermediate conformational states in the B↔Z transitions of poly(dG?dC) · poly(dG?dC)

Time-dependent uv absorption and CD spectrum changes in salt-induced conformational B → Z and Z → B transitions of poly(dG? dC) · poly(dG? dC) were measured. This polynucleotide does not convert directly from a right-handed double-helical B form to a left-handed double-helical Z form, but goes through an intermediate, B* form, with the B → B* transition proceeding nearly instantaneously, and then transforms gradually to the Z form. Uv absorption spectra of these B and B* forms are nearly identical, but their CD spectra are quite different. The CD spectrum of the B* form is identical with that obtained for DNA in high salt solutions and is similar to a spectrum which for some time was thought to be a C form. These B and B* forms have the same number of base pairs per turn [Sprecher, C.A., Baase, W.A. & Johnson, Jr., W.C. (1979) Biopolymers 18 , 1009–1019]. Kinetic measurements showed that uv absorption and CD intensities at fixed wavelengths do not change in a simple exponential curve. However, both the uv absorption spectrum change in the B → Z transition and the CD spectrum change in the B* → Z transition, respectively, have isosbestic points. In the B → Z transition, no hyperchromicity can be observed. These results suggest that this B* form unfolding or premelting process is a rate-determining step in the B* → Z transition and makes it easy for the unfolded or premelted polynucleotide to almost immediately fold into the Z form. The double helix does not dissociate into single strands and transforms from the B* form to the Z form point-by-point along the chain in a soliton-like manner of with a small amount of open states in which the bases are unpaired. Also, in the Z → B transition, the polynucleotide does not convert directly from the Z to the B form, but goes through a B*-like form. In this transition, the uv-absorption spectra also have an isosbestic point. The reaction velocity in the Z → B transition is much faster than that in the B → Z transition. Possibly, the positive CD band between 265 and 310 nm in the B form comes from a n-π* transition due to an interaction of the bases with sugarphosphate groups.     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

Delta V0 of the Na(+)-induced B-Z transition of poly[d(G-C)] is positive

The equilibrium between the right- and left-handed conformations of poly[d(G-C)] in aqueous NaCl shifts towards the right-handed (B) form with increasing pressure. The optical density at 290 and 260 nm was determined at 50 and 180 MPa for solutions in which approximately equal amounts of the two conformations were present at 0.1 MPa (atmospheric pressure). Interpretation of the observed changes in terms of a two-state unimolecular reaction mechanism results in an average molar reaction volume (delta V0) equal to 26 cm3 mol-1 at 22 degrees C; that is, the partial molar volume of B form poly[d(G-C)] is smaller than that of the left-handed (Z) form. Based upon the thermodynamics of ion-pair formation in polar solvents, it is proposed that the positive delta V0 reflects a favorable entropy change for the reaction toward the Z conformation. The larger entropy change of the Z form may derive from the release of water molecules from the hydration spheres of the cation and the poly[d(G-C)] due to the formation of ionic interactions with the Z conformer. The delta V0 of the transition is similar in sign and magnitude to the calculated molar volume change of the interaction of Na+ with H2PO4- in water.