Arrangements of human telomere DNA quadruplex in physiologically relevant K+ solutions.

The arrangement of the human telomeric quadruplex in physiologically relevant conditions has not yet been unambiguously determined. Our spectroscopic results suggest that the core quadruplex sequence G(3)(TTAG(3))(3) forms an antiparallel quadruplex of the same basket type in solution containing either K(+) or Na(+) ions. Analogous sequences extended by flanking nucleotides form a mixture of the antiparallel and hybrid (3 + 1) quadruplexes in K(+)-containing solutions. We, however, show that long telomeric DNA behaves in the same way as the basic G(3)(TTAG(3))(3) motif. Both G(3)(TTAG(3))(3) and long telomeric DNA are also able to adopt the (3 + 1) quadruplex structure: Molecular crowding conditions, simulated here by ethanol, induced a slow transition of the K(+)-stabilized quadruplex into the hybrid quadruplex structure and then into a parallel quadruplex arrangement at increased temperatures. Most importantly, we demonstrate that the same transitions can be induced even in aqueous, K(+)-containing solution by increasing the DNA concentration. This is why distinct quadruplex structures were detected for AG(3)(TTAG(3))(3) by X-ray, nuclear magnetic resonance and circular dichrosim spectroscopy: Depending on DNA concentration, the human telomeric DNA can adopt the antiparallel quadruplex, the (3 + 1) structure, or the parallel quadruplex in physiologically relevant concentrations of K(+) ions.

The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH.

UV absorption and CD spectroscopy, along with polyacrylamide gel electrophoresis, were used to study conformational properties of DNA fragments containing the trinucleotide repeat (GCC)(n) (n = 4, 8 or 16), whose expansion is correlated with the fragile X chromosome syndrome. We have found that the conformational spectrum of the (GCC)(n) strand is wider than has been shown so far. (GCC)(n) strands adopt the hairpin described in the literature under a wide range of salt concentrations, but only at alkaline (>7.5) pH values. However, at neutral and slightly acid pH (GCC)(4) and (GCC)(8) strands homodimerize. Our data suggest that the homodimer is a bimolecular tetraplex formed by two parallel-oriented hairpins held together by hemi-protonated intermolecular C.C(+) pairs. The (GCC)(16) strand forms the same tetraplex intramolecularly. We further show that below pH 5 (GCC)(n) strands generate intercalated cytosine tetraplexes, whose molecularity depends on DNA strand length. They are tetramolecular with (GCC)(4), bimolecular with (GCC)(8) and monomolecular with (GCC)(16). i-Tetraplex formation is a complex and slow process. The neutral tetraplex, on the other hand, arises with fast kinetics under physiological conditions. Thus it is a conformational alternative of the (GCC)(n) strand duplex with a complementary (GGC)(n) strand.

Aqueous trifluorethanol solutions simulate the environment of DNA in the crystalline state.

We took 28 fragments of DNA whose crystal structures were known and used CD spectroscopy to search for conditions stabilising the crystal structures in solution. All 28 fragments switched into their crystal structures in 60-80% aqueous trifluorethanol (TFE) to indicate that the crystals affected the conformation of DNA like the concentrated TFE. The fragments crystallising in the B-form also underwent cooperative TFE-induced changes that took place within the wide family of B-form structures, suggesting that the aqueous and crystal B-forms differed as well. Spermine and magnesium or calcium cations, which were contained in the crystallisation buffers, promoted or suppressed the TFE-induced changes of several fragments to indicate that the crystallisation agents can decide which of the possible structures is adopted by the DNA fragment in the crystal.

Changes in properties of DNA caused by gamma and ultraviolet radiation. Dependence of conformational changes on the chemical nature of the damage.

Changes in the pulse-polarographic behaviour and circular dichroism spectra of DNA were investigated after gamma and ultraviolet irradiations and after degradation by DNAse I. It was found that moderate doses of radiation cause local conformational changes in the double helix, which are dependent on the chemical nature of the damage. Only the accumulation of structural changes after high doses of the radiations or after extensive enzymic treatment may cause formation of single-stranded regions in DNA.

Conformations of alternating purine-pyrimidine DNAs in high-CsF solutions and their reversal by dipyrandium, ethidium and high temperature.

Chiroptical properties of synthetic DNAs with alternating purine-pyrimidine sequences were studied in high-CsF solutions. It was found that this salt transformed all the DNAs to non-canonical conformations like Z or X, which show striking negative bands in the long wavelength part of the CD spectra. The negative bands were reversed upon interaction with dipyrandium, ethidium and at high temperature.

(CGA)(4): parallel, anti-parallel, right-handed and left-handed homoduplexes of a trinucleotide repeat DNA.

Conformational properties of microsatellite DNA regions are the probable reason of their expansions in genomes which lead to serious genetic diseases in some cases. Using CD spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we study in this paper conformational properties of (CGA)(4) and compare them with those of (CAG)(4) - a related repeat, connected with Huntington's disease. We show that (CGA)(4) can adopt several distinct conformations in solution. Around neutral pH it forms a parallel-stranded homoduplex containing C(+).C, G.G, and A.A base pairs. Under the same conditions (CAG)(4) forms a hairpin. At slightly alkaline pH values and low ionic strength, (CGA)(4) also folded into a hairpin which transformed into a bimolecular anti-parallel homoduplex at increasing salt concentrations. The duplex easily isomerized into left-handed Z-DNA, implying that the mismatched adenines between G.C pairs facilitate rather than hinder the B-Z transition. No similar changes took place with (CAG)(4). Thus, the conformational repertoire of (CGA)(4) includes parallel, anti-parallel, right-handed, and left-handed homoduplexes. In contrast, (CAG)(4) invariably adopts only a single conformation, namely the very stable hairpin.

Sequence-dependent changes in the chiroptical properties of DNA upon interaction with dipyrandium.

Interaction of dipyrandium with DNA and its dependence on the base sequence was studied using circular dichroism. It was found that calf thymus DNA and polynucleotide duplexes with alternating purine-pyrimidine sequences containing GC basepairs underwent similar alterations in the chiroptical properties upon binding of dipyrandium. The alterations suggest that these DNAs have similar B-type structures which may kink at the dipyrandium binding sites. On the other hand, poly(dA-dT).poly(dA-dT) and especially poly(dA-dU).poly(dA-dU) exhibit some features of A-type structure. Poly(dA-dT).poly(dA-dT) changes its chiroptical properties little when complexed with dipyrandium, as if it contained some type of kinks as equilibrium structural elements.

Circular dichroism anisotrophy of DNA with different modifications at N7 of guanine.

The complexex DNA-Ag1+, DNA-Cu1+, protonated DNA and DNA methylated at N7 of guanine were oriented by pumping the solutions through a multicapillary cell in the direction of a light beam. The CD components along the DNA axis, delta epsilon parallel, and normal to it, 2 delta epsilon perpendicular, were calculated from the CD spectra of the oriented samples by the method of Chung and Holzwarth, (1975) J. Mol. Biol. 92, 449--466. It was shown that in most cases, except that of the protonated DNA, the degree of orientation was only slightly less than that for pure DNA. This demonstrated the absence of aggregation and of appreciable denaturation. In all cases the modifications of DNA give rise to a negative component 2 delta epsilon perpendicular, whose magnitude increased as the extent of modification increased. From both the CD spectra of non-oriented samples and the absorption spectra, an inference is drawn that Ag1+ and Cu1+ are attached to the same site as CH3 groups i.e., to the N7 atom of guanine. Proton transfer along the H-bond from the N1 atom of G to the N3 atom of the complementary cytosine is suggested to be a result of the modifications, although the case of H+-DNA may differ from the others. Based on the CD spectra for the anisotropic components, delta epsilon parallel and 2 delta epsilon perpendicular, it is proposed that ligand binding is accompanied by winding of the DNA helix.

Salt-induced conformational transition of poly[d(A-T)] X poly[d(A-T)].

Unique chiroptical properties of poly[d(A-T)] X poly[d(A-T)] observed in CsF solutions (Vorlícková et al., 1980) were specified by circular dichroism, ultraviolet light and 31P nuclear magnetic resonance spectroscopy. It was found that up to a 3 M concentration of the salt, caesium cations induced a gradual rearrangement of the polynucleotide double helix during which the phosphodiester bonds in one sequence changed the geometry and the base stacking decreased. At higher CsF concentrations poly[d(A-T)] X poly[d(A-T)] underwent a transition toward a novel conformation which had phosphodiester bonds in both sequences in considerably different non-B-DNA geometries.

An A-type double helix of DNA having B-type puckering of the deoxyribose rings.

DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(C(n)G(n)) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition.

A-like guanine-guanine stacking in the aqueous DNA duplex of d(GGGGCCCC).

We have used CD spectroscopy, NMR spectroscopy and unrestrained molecular dynamics to study conformational properties of a DNA duplex formed by the self-complementary octamer d(GGGGCCCC). Its unusual CD spectrum contains features indicating A-like stacking of half of the bases, whereas the other half stack in a B-like fashion. Unrestrained molecular dynamics simulations converged to a stable B-like double-helix of d(GGGGCCCC). However, the double-helix contained a central hole whose size was half of that occurring in structure A. In the canonical structure B, the hole does not exist at all because the base-pairs cross the double-helix centre. The cytosine bases were stacked in the duplex of d(GGGGCCCC) as in structure B, while stacking of the guanine bases displayed features characteristic for structure A. NMR spectroscopy revealed that the A-like guanine-guanine stacking was accompanied by an increased tendency of the deoxyribose rings attached to the guanine bases to be puckered in an A-like fashion. Otherwise, the duplex of d(GGGGCCCC) showed no clash, no bend and no other significant deviation from structure B. The present analysis demonstrates a remarkable propensity of the guanine runs to stack in an A-like fashion even within the B-DNA framework. This property explains why the oligo(dG). oligo(dC) tracts switch into structure A so easily. Secondly, this property may influence replication, because structure A is replicated more faithfully than structure B. Thirdly, the oligo(dG) runs might have played an important role in early evolution, when DNA took on functions that originally evolved on RNA. Fourthly, the present study extends the vocabulary of DNA secondary structures by the heteronomous duplex of d(GGGGCCCC) in which the B-like strand of oligo(dC) is bound to the A-like strand of oligo(dG).

Divalent cations are not required for the stability of the low-salt Z-DNA conformation in poly(dG-ethyl5dC).

It is demonstrated that poly(dG-ethyl5dC) adopts Z form in low-salt solution like poly(dG-methyl5dC). Its existence is, however, not contingent on the presence of divalent cations in the polynucleotide solution. The Z form is transformed into B form below room temperature. The arising B form cannot be transformed back into Z form by millimolar MgCl2 concentrations. On the contrary, the addition of MgCl2 at room temperature converts the low-salt Z form of poly(dG-ethyl5dC) into B form. It follows from the results that Z form is a stable DNA conformation not only at high but even at low ionic strengths.

Conformational variability of poly(dA-dT).poly(dA-dT) and some other deoxyribonucleic acids includes a novel type of double helix.

The article reviews data indicating that poly(dA-dT).poly(dA-dT) is able of adopting three distinct double helical structures in solution, of which only the A form conforms to classical notions. The other two structures have dinucleotides as double helical repeats. At low salt concentrations poly(dA-dT).poly(dA-dT) adopts a B-type alternating conformation which is exceptionally variable. Its architecture can gradually move in the limits demarcated by the CD spectra with inverted long wavelength CD bands and the 31P NMR spectra with a very low and a 0.6 ppm separation of two resonances. Contrary to Z-DNA, the 31P NMR spectrum of the limiting alternating B conformation of poly(dA-dT).poly(dA-dT) is characterized by an upfield shift of one resonance. We attribute the exceptional conformational flexibility of the alternating B conformation to the unequal tendency of bases in the dA-dT and dT-dA steps to stack. However, by assuming the limiting alternating B conformation, the variability of the synthetic DNA is not exhausted. Specific agents make it isomerize into another conformation by a fast, two-state mechanism, which is reflected by a further deepening of the negative long wavelength CD band and a downfield shift of the 31P NMR resonance of poly(dA-dT).poly(dA-dT) that was constant in the course of the gradual alterations of the alternating B conformation. These changes are, however, qualitatively different from the way poly(dG-dC).poly(dG-dC) behaves in the course of the B-Z isomerization. Poly(dG-dC).poly(dG-dC) displays purine-pyrimidine (dGpdC) resonance in the characteristic downfield position, while the downfield resonance of poly(dA-dT).poly(dA-dT) belongs to the pyrimidine-purine (dTpdA) phosphodiester linkages. Consequently, phosphodiester linkages in the purine-pyrimidine steps play a similar role in the appearance of the Z form to the pyrimidine-purine phosphodiesters in the course of the isomerization of poly(dA-dT).poly(dA-dT). This excludes that the high-salt structures of poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) are members of the same conformational family. We call the high-salt conformation of poly(dA-dT).poly(dA-dT) X-DNA. It furthermore follows from the review that synthetic molecules of DNA with alternating purine-pyrimidine sequences of bases can adopt either the Z form or the X form, or even both, depending on the environmental conditions. This introduces a new dimension into the DNA double helix conformational variability. The possible biological relevance of the X form is suggested by experiments with linear molecules of natural DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular dichroism studies of salt- and alcohol- induced conformational changes in cyanophage S-2L DNA which contains amino 2 adenine instead of adenine.

DNA molecules containing AT pairs exhibit cesium cation specific conformational behavior. This specificity is shown to be cancelled with the title DNA, which not only concerns its conformational alterations in high-salt aqueous solutions but also the B-to-A transition induced by ethanol. S-2L DNA easily adopts the A-conformation in the presence of millimolar concentrations of CsCl which completely destabilize the A-conformation in calf thymus DNA. The present results demonstrate that the specific effects of cesium cations on DNA are connected with their binding to the AT pairs in the DNA minor groove.

Alkyl substituent in place of the thymine methyl group controls the A-X conformational bimorphism in poly(dA-dT).

Circular dichroism studies of a family of poly(dA-y5dU) polynucleotides (y = H, methyl, ethyl, propyl, butyl or pentyl) were conducted in water-alcohol solutions containing sodium or cesium counterions. The polynucleotides denatured or adopted A- or X-DNA double helices depending on the concentration and type of alcohol, type of counterions and the length of the aliphatic substituent in place of the thymine methyl group. Short aliphatic substituents and sodium cations favored A-DNA while long aliphatic substituents and cesium cations promoted X-DNA. This study demonstrates delicacy of the conformational equilibrium of poly(dA-dT) between the A- and X-DNA double helices which depends on both intramolecular and intermolecular factors.

Probing conformational isomerizations of double-stranded poly(dA-dT) by a substitution of minor amounts of the thymine methyls with bulky hydrophobic isopropyl groups.

We probed conformational polymorphism of a synthetic DNA poly(dA-dT) by introducing various small amounts of bulky spherical hydrophobic isopropyl groups into the polynucleotide primary structure. For this purpose, three mixed copolymers of poly(dA-dT,ip5dU) were synthesized in which 2.6%, 8.6% or 14.2% of the polynucleotide pyrimidine bases had the isopropyl group in position 5. The isopropyls made the formation of both A-form and X-form incomplete, and this effect increased with the increasing isopropyl amount in the polynucleotide. However, the polynucleotide isomerization into the A-form was hindered by the isopropyls while the isomerization into the X-form was rather promoted. This observation indicates that, unlike the A-form, the X-form has the base pairs shifted towards the double helix major groove. Z-form was also promoted by the lowest concentration of the isopropyl groups while the most isopropylated poly(dA-dT) aggregated under the Z-form inducing conditions.

Unusual contribution of 2-aminoadenine to the thermostability of DNA.

The poly(dA-dU) and poly(dI-dC) duplexes have very similar thermostabilities (Tm). This similarity extends also to the pyrimidine 5-methyl group-containing poly(dA-dT) and poly(dI-m5dC). The differences between chemical structures of the A:U and I:C or the A:T and I:m5C base-pairs seem to be unimportant for the thermostability of the DNA. However, on the insertion of an amino group into position 2 of the purines the similarities disappear. Thermostabilities of poly(n2dA-dU) and poly(dG-dC) as well as the poly(n2dA-dT) and poly(dG-m5dC) are radically different. This is also the case with their other 5-substituted pyrimidine-containing derivatives, the 5-ethyl, 5-n-butyl and 5-bromo analogues. The G:C-based polynucleotides are more stable by an average of 40 degrees C than the n2A.U-based ones. Poly(dA,n2dA-dT)-s containing various proportions of A and n2A as well as the natural DNA of S-2L cyanophage that contains n2A bases instead of A were also studied. It was found that dependence of Tm on the n2A-content was non-linear and that the lower Tm is not the consequence of a particular nucleotide sequence. The possible structural reasons for the lower thermostabilization of these B-DNAs by the n2A:T base-pair as compared to the G:C are discussed.

UV light-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs.

Alkaline agarose gel electrophoresis was used to detect UV-induced crosslinking of the strands of poly(dA-dT) and related alternating purine-pyrimidine DNAs in solutions stabilizing various polynucleotide conformations. Strands of the B-form and A-form of poly(dA-dT) were not crosslinked but a UV dose-dependent retarded species appeared in the denaturing gels in parallel with the polynucleotide isomerization into the unusual X-form. Most other polynucleotides adopting the X-form were crosslinked as well. The exceptions include the X-forms of poly(dA-butyl5dU) and poly(dA-pentyl5dU) whose strands do not crosslink because the long exocyclic substituents attached to uracil make the photodimerization impossible. Strands of poly(amino2dA-dT) and poly(dA, amino2dA-dT), the latter polynucleotide containing roughly equal amounts of amino2adenine and adenine, also do not crosslink upon UV irradiation because they isomerize into an A-like conformation which is different from the X-form of poly (dA-dT). In contrast, strands of the mixed copolymers of poly(dA, amino2dA-dT) containing low amino2adenine contents are crosslinked upon UV irradiation, in accordance with the observation that they isomerize into the X-form.

Replication, transcription and nuclease digestion of the unusual X-DNA double helix of poly(amino2dA-dT).

The alternating copolymer poly(amino2dA-dT) isomerizes into the unusual X-DNA double helix at low-salt aqueous conditions (Vorlicková et al., J. Biomolec. Struct. Dyn. 6, 503-510 (1988)). This observation allowed us to start studies on how the X-DNA is recognized, copied and hydrolyzed by various enzymes. In the present paper X-DNA replication, transcription and digestion by various polymerases and nucleases, respectively, are examined and compared to appropriate controls. It is found that X-DNA is a poor primer-template for DNA synthesis by the E. coli Klenow DNA polymerase (12% of the activity observed with B-DNA), the Micrococcus luteus DNA polymerase I (25%) and the AMV reverse transcriptase (51%). In contrast, X-DNA is a better template by 74% than B-DNA for calf thymus DNA polymerase alpha. For transcription by E. coli RNA polymerase enzyme poly(amino2dA-dT) did not serve as a template at all in either B or X conformation. Poly(amino2dA-dT) in its B form proved to be much more stable than poly(dA-dT) against hydrolysis by pancreatic DNase and snake venom phosphodiesterase. Formation of the X conformation in poly(amino2dA-dT) decreased this large difference in nuclease stability.