B,Z Conformations and Mechanism of the Z-B-Coil Transitions of the Self-Complementary Deoxy-hexanucleotide d(C-G-m5C-G-C-G) by 1H-NMR and CD Spectroscopy

Abstract

The helical structures of d(C-G-m⁵C-G-C-G) were studied in aqueous solution at various salt concentrations and temperatures by CD and ¹H-NMR spectroscopy. At room temperature only the B form is observed in 0.1 M NaCl whereas the B and Z forms are simultaneously present in 1.8 M NaCl. At high salt concentration (4 M NaCl) the Z form is largely predominant (> 95%). The Z form proton resonances were assigned by using the polarisation transfer method (between B and Z at 1.8 M NaCl) and by proton-proton decoupling (at high salt concentration).

The Z-B-Coil transitions were studied as a function of temperature with the 1.8 M NaCl solution. At high temperature (95°C) only the coil form (S) is present. Below 55°C the coil proportion is negligible, and the B-Z exchange is slow. The disappearance of the coil gives rise at first to the B form and on lowering the temperature the Z proportion increases to the detriment of the B form. Proton linewidth, relaxation and polarisation transfer studies confirm the conclusion in the previous report on d(m⁵C-G-C-G-m⁵C-G) (Tran-Dinh et al Biochemistry 1984 in the press) that Z exchanges only with B whereas the latter also exchanges with S,Z ? B ? S. The present data show that even at high salt concentration where only the Z form of d(C-G-m⁵C-G-C-G) is observed the Z-S transition also passes through the B form as an intermediate stage. The B-Z transition takes place when the Watson-Crick hydrogen bonds are firmly maintained and is greatly favoured when there are three hydrogen bonds between the base-pairs.     

1H NMR and circular dichroism studies of the B and Z conformations of the self-complementary deoxyhexanucleotide d(m5C-G-C-G-m5-C-G): mechanism of the Z-B-coil transitions

The double-helical conformations of d(m5-C-G-C-G-m5-C-G) in aqueous solution were studied by circular dichroism and 1H NMR spectroscopy. In 0.1 M NaCl, only the B form is detected whereas the Z form is strongly predominant in 3 M NaCl. In the presence of 2 M NaCl, two resonance signals corresponding to the B and Z duplexes were observed for each proton below 50 degrees C, indicating a slow exchange between B and Z. However, the B-Z exchange becomes intermediate or fast in the 55-80 degrees C temperature interval. By contrast the exchange between B helix and single-stranded (or coil) forms is much faster for the same temperature conditions. The Z form is only detectable when the coil form is practically absent. With decreasing temperature the B form decreases in favor of the Z form. From proton line-width measurements under various experimental conditions, it was also shown that Z exchanges only with B, while the latter also exchanges with the single-stranded form (S): Z in equilibrium B in equilibrium S. The enthalpy value is about 8 +/- 1 kcal/mol for the B-Z transition and about 40 +/- 2 kcal/mol for the B-S dissociation (2 M NaCl solution). The activation energy is about 47 +/- 2 kcal/mol for the Z----B and 39 +/- 2 kcal/mol for the B----Z reaction. Very good agreement between the experimental results and computed data (based on the above kinetic reaction model) was found for the B, Z, and coil proportions. The B-Z transition of methylated d(C-G)n oligomers is only possible when the Watson-Crick hydrogen bonds between the CG base pairs are firmly maintained; otherwise, the transformation from B to Z would not occur, and B-S dissociation would take place instead.     

Thermodynamic behaviour of the heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG) forming B and Z hairpins in aqueous solution.

UV and CD data of the partially self-complementary heptadecadeoxynucleotide d(CGCGCGTTTTTCGCGCG), obtained as a function of temperature, salt and strand concentration, show that: at low NaCl and strand concentration the oligomer exhibits, on increasing the temperature, a biphasic thermal profile which is indicative of two structural transitions, from dimeric duplex to hairpin and from hairpin to coil; the loop stabilizes enthalpically both B and Z hairpin structures with respect to the corresponding unconstrained hexamer d(CGCGCG) by a few Kcal/mol; the oligomer undergoes a B-Z transition which appears to be complete, at 0 degree C, when induced by NaClO4; by contrast the B-Z transition induced by NaCl does not reach completeness even at salt saturation. The independence of the denaturation temperature, at high salt conditions, on the oligomer concentration indicates that the Z structure is present also in the hairpin.     

Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.     

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.     

The length of a junction between the B and Z conformations in DNA is three base pairs or less

Recently it has been suggested that double-helical complexes formed between the DNA sequences (CG)n(A)m and their conjugates, (T)m(CG)n, would be candidates for the formation of a B-Z junction in aqueous solution at high salt concentrations [Peticolas et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 2579-2583]. The junction was predicted to occur between a B-type helix in the d(A)m.d(T)m section and a Z-type helix in the self-complementary (CG)n.(CG)n sequence. In this paper we report Raman experiments on the deoxyoligonucleotides d(CGCGCGCGCGCGAAAAA) and d(CGCGCGAAAAA) and their complements. It is found the latter compound cannot be induced into the Z form in saturated salt solution but that the former sequence goes into a B-Z junction at 5.5 M salt. From a comparison of the relative intensity of the Raman conformational marker bands for B and Z DNA for both the A-T and C-G base pairs, it is shown that in 5.5 M NaCl solution none of the A-T base pairs are in the Z form, but nine of the C-G base pairs are in the Z form. The remaining three C-G base pairs are either in the junction or in the B form. Thus, the junction is formed from three or less C-G base pairs. If the solution is made 95 microM with NiCl2, then the entire duplex goes into the Z form and the Raman bands of the adenine are completely changed into those of the Z form.(ABSTRACT TRUNCATED AT 250 WORDS)     

An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C-G-m5C-G-A-G-m5C-G) in solution. The B-duplex and hairpin forms

By means of one- and two-dimensional NMR spectroscopy the solution structures of the partly self-complementary octamer d(m5C-G-m5C-G-A-G-m5C-G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random-coil form. For the hairpin/random-coil equilibrium a Tm of 329 K and a delta H degree of -121 kJ.mol-1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K (1 M DNA) and a delta H degree of -285 kJ.mol duplex-1 were derived. The thermodynamic data of the hairpin-coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939-963]. It is demonstrated that the dimer structure exhibits B-DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton-proton coupling data. It is shown that the base-pair formation of the G x A mismatches is anti-anti. A comparison of 1H and 31P chemical-shift data of the title compound with those of a well-characterized B-DNA structure reveals large differences in the dm5C(3)-dG(4)-dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G x A mismatches. Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z-DNA structure was observed. It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C-G-m5C-G-T-G-m5C-G). An energy-minimized model of the hairpin form is given.     

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.     

B-A and B-Z transitions in deoxyoligoduplexes containing 4- and 5- methylcytosine

Self-complementary oligodeoxynucleotides: GGACCCGGGTCC, GGA4mCCCGGGTCC, GGA5mCCCGGGTCC, CGCGCGCG, CG4mCGCGCG, CG5mCGCGCG were synthetized to study the contribution of methyl groups into the energetics of the three known cooperative transitions in DNA: helix-coil, B-A and B-Z With the use of circular dichroism and absorbtion methods the profiles of the above transitions were obtained by variation of temperature (helix-coil), trifluoroethanol fraction (B-A), NaCl and trifluorethanol contents (B-Z). On the basis of the transition widths and shifts of the transition points due to the methylations the energetics of the methyl groups was estimated. 5mC stabilizes the B form relatively the A form by 0.33 kcal/mol; while 4mC by 0.5 kcal/mol. In the B-Z transition 5 mC stabilizes the Z form by 0.28 kcal/mol relatively the B form; 4mC stabilizes also the Z form although by 0.14 kcal/mol only. Thus, these naturally occurring modifications could modulate substantially the ability of a DNA piece to shift into the A or Z form.     

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.     

Effect of P-chirality of internucleotide bonds on B-Z conversion of stereodefined self-complementary phosphorothioate oligonucleotides of the [PS]-d(CG)4 and [PS]-d(GC)4 series

Diastereomerically pure, partially modified (in selected positions) or fully modified phosphorothioate oligomers of the [PS]-d(CG)(4) and [PS]-d(GC)(4) series were investigated with respect to their ability to adopt the left-handed conformation at high sodium chloride concentration. NaCl induces the B-Z transition of [All-S(P)R(P)-PS]-d(CG)(4) with a midpoint of transition at ca. 2 M, which is approximately 1 M less than for unmodified d(CG)(4). Also, [All-R(P)S(P)-PS]-d(GC)(4) at 5 M NaCl converts to the Z form to the extent of ca. 55%, while the unmodified d(GC)(4) counterpart does not convert at all. This enhanced ability of stereodefined phosphorothioate oligomers to adopt the Z conformation is discussed in terms of already known structural factors (hydrogen bonding and water bridges) facilitating the B-Z transition, identified for unmodified d(CG)(n) oligonucleotides. By CD spectroscopy, the [All-S(P)-PS]-d(CG)(4) oligomer at a NaCl concentration higher than 0.01 M adopts a unique conformation as assessed from the presence of an additional negative band centered at 282 nm.     

1H-NMR study of the interaction of daunomycin with B-DNA helices of methylated oligodeoxynucleotides.

The interaction of daunomycin with B-DNA double helices of several methylated deoxynucleotides, d(C-G-m5C-G), d(m5C-G-C-G), d(C-G-m5C-G-C-G) and d(m5C-G-C-G-m5C-G) in solution was investigated by 1H-NMR spectroscopy at 500 MHz. At low temperature (t less than 20 degrees C for the tetramer and t less than 40 degrees C for the hexamers), several daunomycin-DNA complexes were observed in slow exchange with the drug-free DNA duplexes. The presence of daunomycin in a self-complementary double helix cancels the conformational symmetry of the two strands; the proton signals can split into several others owing to the difference between free and intercalated duplexes and to the many possible intercalation sites in a duplex (three for a tetramer, five for an hexamer). A model relating the chemical shifts of splitted proton signals to the various intercalated duplex conformations was given. The results show that one daunomycin molecule is associated with one duplex and that it can enter any intercalation site with equal probability; no side-effects were observed even for very short helices (of a tetramer). In the case of d(C-G-m5C-G) the association constant and the dissociation and association rates of the intercalated complex were evaluated.     

Z helix-coil transition of d(C-Br8G-C-G-C-Br8G) studied by CD, 1H-NMR and IR spectroscopies

The conformation of d(C-Br8G-C-G-C-Br8G) in aqueous solution was studied by CD and 1H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)3 oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of d(C-Br8G-C-G-C-Br8G) demonstrating the duplex formation were observed up to 60 degrees C. It is interesting to note that the significant highfield shifts of the dC H5" exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)3 containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)3 oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br8G-C-G-C-Br8G) is obtained directly from the coil form. However, IR data suggest that in the Z form of d(C-Br8G-C-G-C-Br8G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)3 crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)3 are compared to those of the B helix-coil transition observed for methylated d(C-G)3 in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.     

Hairpin and duplex formation of the DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) in solution. An NMR study.

The partly self-complementary DNA octamer d(m5C-G-m5C-G-T-G-m5C-G) was investigated by NMR spectroscopy in solution. It is demonstrated that this peculiar DNA fragment, under suitable conditions of concentration, salt and temperature, exclusively prefers to adopt a monomeric hairpin form with a stem of three Watson-Crick type base pairs and a loop of two residues. At high single strand concentration (8 mM DNA) and low temperature (i.e. below 295 K) the hairpin occurs in slow equilibrium with a B-dimer structure. At high ionic strength (greater than or equal to 100 mM Na+) and/or in the presence of methanol a third species appears, which is assigned to a Z-like dimer. In the B form, as well as in the Z dimer, the two central base pairs form G.T wobble pairs with the bases as major tautomers.     

B-Z cooperativity and kinetics of poly(dG-m5dC) are controlled by an unfavorable B-Z interface energy

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m5dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl2, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl2 in 50 mM NaCl, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 +/- 0.3 kcal/mol and is length independent. Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 +/- 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event. Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

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.     

Influence of dA X dT and d(2aminoA) X dT base pairs on the B in equilibrium Z transition of DNA fragments. 1H-NMR study of d(C-G-C-A-m5C-G-T-G-m5C-G), d(m5C-G-C-A-m5C-G-T-G-C-G) and d(C-2aminoA-C-G-T-G)

The Helical structures of d(C-G-C-A-m5C-G-T-G-m5C-G), d(m5C-G-C-A-m5C-G-T-G-C-G) and d(C-2aminoA-C-G-T-G) were studied in aqueous solution at various salt concentrations and temperatures by 1H-NMR spectroscopy. In 0.1 M NaCl solution only the B form was evidenced for these DNA fragments whereas in 4 M NaCl both B and Z forms, in slow exchange on the NMR time scale, were observed. Under these conditions the Z form accounted for less than 60% of the decamer conformation; conversely d(C-G)3 hexamers containing methylated cytidines were predominantly in the Z form (greater than 90%) [Tran-Dinh et al. (1984) Biochemistry 23, 1362; Cavaillès et al. (1984) J. Biomol. Struct. Dyn. 1, 1347-1371]. On the other hand, d(C-2aminoA-C-G-T-G) in which the d(2aminoA) X dT base pair forms three hydrogen bonds, was found to adopt the Z conformation in 4M NaCl solution which was not the case for d(C-A-C-G-T-G) (unpublished results). The present study shows that the B in equilibrium Z transition in solution is highly sequence-dependent and that correlation exists between the stability of the duplexes (essentially governed by the number of hydrogen bonds between complementary bases) and their ability to adopt the Z conformation.     

Sequence dependence of the B to Z transition in crystals and aqueous NaCl solutions for deoxyoligonucleotides containing all four canonical DNA bases

A laser Raman study has been made on the conformation of a series of self-complementary octameric deoxynucleotides that contain all four canonical deoxynucleotide bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] in order to determine which sequences will crystallize in the Z form and which sequences will go into the Z form in aqueous solution at high salt concentrations (4-6 M NaCl). All four octadeoxynucleotides, d(TGCGCGCA) (I), d(CACGCGTG) (II), d(CGTGCACG) (III), and d(CGCATGCG) (IV), have been crystallized from low-salt solutions. The Raman spectra of microcrystals show that I, II, and IV crystallize in a rigorous Z form while III crystallizes in the B form. Sequences I and II go into a Z form in 4-6 M NaCl solution at 0 degrees C while sequences III and IV remain in the B form in 6 M salt. There are substantial differences in the Raman spectra of oligonucleotides in the Z form found in the crystal and in high-salt solutions. The Raman spectra of the Z forms in 6 M NaCl solution at 0 degrees C are not linear combinations of the Raman spectra of the complete Z form in the crystal and the complete B form in low-salt solutions. The terminal residues of these oligomers do not appear to be in a strict Z form. A detailed analysis of the ring puckers and syn/anti conformation for all of the residues both in solution and in the crystal has been made.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

B--Z conformational transition in d(CGCGCGTTAATT), d(CGCGCGTTAA) and d(CGCGCGTT)

Conformational studies on three DNA-oligomers (d(CGCGCGTTAATT), d(CGCGTTAA) and d(CGCGCGTT) in solution by circular dichroism spectroscopy are reported. In low salt solution, all three DNA oligomers exhibit a characteristic B-conformation. However, under the influence of high salt concentration i.e. 5M NaCl, the octamer d(CGCGCGTT) exhibits 'A' conformation whereas the decamer and dodecamer retain B-conformation. On addition of millimolar amount of NiCl2 to the 5M NaCl, solution of oligodeoxynucleotides a B-Z transition is observed in octamer, decamer and dodecamer. However, NiCl2 titrations show that mid point of transition for dodecamer is at 2.25 mM, for decamer is at 13 mM NiCl2 and for octamer is 17 mM at NiCl2. In 60% alcohol all three oligonucleotides remain in the B-conformation. The melting temperatures of oligonucleotides at various salt concentration are also reported. Thermodynamic parameters calculated by melting profile using a two state model show that dodecamer and decamer are most stable in their 5M NaCl, B-form. However, octamer is more stable in its Z form than that of its 'A' form.