A pH-dependent Structural Transition in the Homopurine-homopyrimidine Tract in Superhelical DNA

Abstract

We have inserted the 509-bp-long fragment of sea urchin P. miliaris histone gene spacer region into plasmid pUC19. The fragment contains the 60-bp-long homopurine-homopyrimidine tract that is known to be hypersensitive to the S1 endonuclease. Using two-dimensional gel electrophoresis we have observed a sharp structural transition in the insert with increasing DNA superhelicity. As in the cases of cruciform and Z form formation, the observed transition partly relaxes the superhelical stress. In contrast with the other two well documented transitions, the observed transition strongly depends on pH. At pH7 and above the transition occurs at negative superhelicities exceeding the physiological range (σ>0.08). For pH6 the transition occurs at ?σ = 0.055, whereas for pH4.3 it takes place at ?σ = 0.001. A comprehensive analysis of the obtained data has made it possible to define the nature of the observed transition. We conclude that under superhelical stress or/and at low pH homopurine- homopyrimidine tracts adopt a novel spatial structure called the H form.     

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.     

Fluctuations in superhelical DNA.

The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.     

A structural transition in d(AT)n.d(AT)n inserts within superhelical DNA

We have constructed plasmids carrying d(AT)n.d(AT)n inserts of different lengths. Two-dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease S1, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)n.d(AT)n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

Effect of length, supercoiling, and pH on intramolecular triplex formation. Multiple conformers at pur.pyr mirror repeats

The conformations adopted by five oligopurine.oligopyrimidine (pur.pyr) inserts of various lengths and sequence repeats in recombinant plasmids were evaluated as a function of pH and negative super-helicaldensity. Patterns of chemical reactivity (OsO4 and diethylpyrocarbonate) indicate that long (greater than 36 base pairs) pur.pyr segments can adopt intramolecular triplexes and that increasing the length of the pur.pyr tract reduces the dependence on low pH for structure formation, such that (GA)37 adopts an intramolecular triplex under moderate levels of negative superhelical stress (-sigma = 0.049) at neutral pH. This demonstrates that long pur.pyr segments, which are abundant in eukaryotic genomes, have the potential to adopt triplexes in vivo. Two-dimensional gel electrophoresis of the plasmids combined with chemical probing indicates that for longer sequences, multiple conformers of the intramolecular triplex exist at low pH. These conformers result from nucleation at various positions on the polypurine stretch, giving rise to different extents of relaxation at the same linking number. In addition, the metal ions Co2+, Mn2+, and Mg2+ have profound effects on the pattern of chemical reactivity displayed by long pur.pyr segments at both neutral and low pH, indicating that quite different structures may form in the presence of divalent metal ions. Thus, the types and extent of unusual structures adopted by long pur.pyr segments are complex and heterogeneous, and are dependent on pH, supercoiling, and the presence of divalent cations.     

Environmental influences on DNA superhelicity. The effect of ionic strength on superhelix conformation in solution

The techniques of small-angle X-ray scattering and analysis that have been developed by the authors are used to investigate the influence of ionic strength on the superhelical conformation of native COP608 plasmid DNA in solution. For salt concentrations below 0.1 M, the superhelicity is partitioned between twisting (Tw) and writhing (Wr) in the ratio delta Tw/Wr = 2. Near the physiological salt concentration, [Na+] = 0.2 M, a co-operative transition is observed in which the pitch angle of the toroidal superhelix is drastically decreased. This results in an almost complete relaxation of writhe. At salt concentrations in excess of the threshold for this transition, the superhelical partitioning occurs in the ratio delta Tw/Wr greater than 25. Energetic considerations support the suggestion that this transition results from co-operative, superhelical B to Z transconformation reactions at susceptible sites. A method is discussed that will enable the direct measurement of this secondary structural transition by means of X-ray scattering.     

Extracellular nucleases of Alteromonas espejiana BAL 31.IV. The single strand-specific deoxyriboendonuclease activity as a probe for regions of altered secondary structure in negatively and positively supercoiled closed circular DNA.

The dependence of the initial rate of introduction of the first single-chain scission (initial nicking rate) into covalently closed circular phage PM2 DNA by the single strand-specific nuclease from Alteromonas espejiana BAL 31 upon the superhelix density (sigma) of the DNA has been examined. The initial nicking rate decreases with decreasing numbers of negative superhelical turns (decreasing values of -sigma), which behavior is characteristic of other single strand-specific nucleases as reported earlier. In contrast to earlier work, the initial nicking rates of closed circular DNAs by the action of the Alteromonas nuclease have been shown to be readily measurable at values of -sigma as low as 0.02. However, even at the elevated concentrations of enzyme and extended digestion periods required to cause nicking at an appreciable rate at near-zero values of sigma, closed circular DNA containing very few superhelical turns (form IO DNA) is not cleaved at a detectable rate. When this DNA is rendered positively supercoiled by ethidium bromide (EtdBr), it is not affected by the nuclease until very high positive values of sigma are attained, at which low rates of cleavage can be detected at elevated enzyme concentrations. The effects of EtdBr on the enzyme activity have been tested and are entirely insufficient to allow the interpretation of zero nicking rates as the result of inhibition of the nuclease activity by the dye. Positively supercoiled DNA is concluded not to contain regions having significant single-stranded character until values of sigma are reached which are very much higher than the values of -sigma for which negatively supercoiled DNAs behave as if they contain unpaired or weakly paired bases.     

Conformational DNA transition in the in vitro torsionally strained chicken beta-globin 5'' region.

A sequence of 86 bp within the 5' region of the adult chicken beta-globin gene was found to undergo a DNA conformational transition at elevated levels of negative superhelical stress (- sigma = 0.068). In vitro chemical DNA modification studies which detect purine hyperreactivity (HR) to the alkylating agent diethyl pyrocarbonate (DEP) have identified this 86 bp long DEP-HR element. The DEP-HR element is composed of small, tandem segments with imperfect purine-pyrimidine alternations. Methylation of cytosines within GCGC sequences of the DEP-HR element facilitates this structural change. The binding of a monoclonal anti-Z-DNA antibody to the element has been revealed by chemical footprinting with DEP. These data suggest that the DEP-HR sequence can undergo a conformational transition to Z-DNA. It is unknown whether the conformational flexibility observed here occurs in vivo.     

Energetics of the strand separation transition in superhelical DNA.

In this paper the values of three free energy parameters governing the superhelical strand separation transition are determined by analysis of available experimental data. These are the free energy, a, needed to initiate a run of separation, the torsional stiffness, C, associated with interstrand winding of the two single strands comprising a separated site and the coefficient, K, of the quadratic free energy associated to residual linking. The experimental data used in this analysis are the locations and relative amounts of strand separation occurring in the pBR322 DNA molecule and the measured residual linking, both evaluated over a range of negative linking differences. The analytic method used treats strand separation as a heteropolymeric, co-operative, two-state transition to a torsionally deformable alternative conformation, which takes place in a circular DNA molecule constrained by the constancy of its linking number. The values determined for these parameters under the experimental conditions (T = 310 K, pH = 7.0, monovalent cation concentration = 0.01 M) are a = 10.84(+/- 0.2) kcal/mol, C = 2.5(+/- 0.3) x 10(-13) erg/rad2 and K = 2350(+/- 80) RT/N, where N is the molecular length in base-pairs. In order to assess the accuracy of the author's theoretical methods, these free energy parameters are incorporated into the analysis of superhelical strand separation in different molecules and under other conditions than those used in their evaluation. First, the temperature dependence of transition is treated, then superhelical strand separation is analyzed in a series of DNA molecules having systematic sequence modifications, and the results of these theoretical analyses are compared with those from experiments. In all molecules, transition is predicted in the range of linking differences where it is seen experimentally. Moreover, it occurs at the specific sequence locations that the analysis predicts, and with approximately the predicted relative amounts of transition at each location. The known sensitivities of this transition to changes of temperature and to small sequence modifications are predicted in a quantitatively precise manner by the theoretical results. The demonstrated high-level precision of these theoretical methods provides a tool for the screening of DNA sequences for sites susceptible to superhelical strand separation, some of which may have regulatory or other biological significance.     

A Structural Transition in d(AT) n ·d(AT) n Inserts within Superhelical DNA

Abstract

We have constructed plasmids carrying d(AT)_n·d(AT)_n inserts of different lengths. Two- dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor for the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease SI, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)_n·d(AT) _n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

Structure of (dG)n.(dC)n under superhelical stress and acid pH

We have recently shown that a (GA)n.(TC)n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2,327 (1985]. Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)n.(dC)n insert to undergo a pH-dependent structural transition similar to that of the (GA)n.(TC)n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3, 667 (1986].     

Kinetic trapping of H-DNA by oligonucleotide binding.

Homopurine-homopyrimidine mirror repeats are known to adopt the H form under acidic pH and/or negative supercoiling. In H-DNA, one half of the purine strand enters the triplex whereas the second half is unstructured and can form duplex with complementary oligonucleotide. However, because the same oligonucleotide can form triplex with the homopurine-homopyrimidine insert, one could expect that oligonucleotide would make H-DNA thermodynamically less favorable, as was claimed by Lyamichev et al. Nucl. Acids Res. 16, 2165-2178 (1988). Now we show that complex between oligonucleotide and H-DNA, formed under conditions favorable for the H-form extrusion, is kinetically trapped in superhelical DNA and remains stable up much higher pH values than H-DNA alone. Experiments on chemical probing show that such complex exists for a plasmid with native superhelical density at pH7. We have also used this approach to demonstrate a pH-dependent structural transition in yeast telomeric sequence, d(CACACCCA)16.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Unusual protonated structure in the homopurine.homopyrimidine tract of supercoiled and linearized plasmids recognized by chemical probes

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively. At pH 6.0 the site-specific modification of the homopurine.homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine.homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine.homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine.homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3,667 (1986).     

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.     

Binding of anti-Z-DNA antibodies to negatively supercoiled SV40 DNA.

The binding of anti-Z-DNA antibody preparations to negatively supercoiled, protein-free SC40 DNA was analyzed. Covalent cross-linking with 0.1% glutaraldehyde followed by DNA restriction endonucleolytic fragmentation and nitrocellulose filtration allowed accurate mapping of antibody binding sites. The critical superhelical density necessary to allow antibody binding was -sigma = 0.056. The major region of antibody-DNA interaction was found within an SV40 segment spanning viral map positions 40 to 474. This region coincides with the nucleosome free region in SV40 minichromosomes and harbours the early and late promoter regions including the SV40 enhancer segment. Although it is unknown whether alternative, non-B-DNA conformations are generated in vivo within SV40 minichromosomes our results emphasize the high degree of DNA structural flexibility that can be realized under negative torsional stress.     

The B-A transition in superhelical DNA.

Relaxation of a DNA superhelical stress due to the B to A transition induced by trifluoroethanol has been studied by assessing the change of DNA orientation in a flow gradient. Using DNAs of different superhelical densities, a decrease in the winding angle during the B----A shift of DNA was found to be 1.5 degrees per base pair in solution. Accepting the winding angle for B-DNA in solution to be 34.1 degrees, that for A-DNA must have a value of 32.6 degrees which agrees with the X-ray data for A-DNA in the condensed state. The date obtained within the B-A transition interval make it possible to conclude that there is an increase in winding at each B/A junction, which is about 5 degrees per one junction.