Formation of noncanonical structures in superhelical DNA. Mutual effects of various transitions

This is a theoretical study of the problem of formation of noncanonical structures, cruciforms in palindromic regions and the Z form in purine--pyrimidine sequences, in negatively supercoiled DNA. If two such regions, one palindromic and one purine--pyrimidine, are present in the same DNA molecule of a finite length, then transitions between the regular B form and noncanonical structures in these regions will experience a considerable mutual influence. This takes place because both noncanonical structures compete for the same superhelix energy. A special attention is paid to the case when the probability of the Z form formation nonmonotonously depends on the superhelix density. Such a situation is shown to be possible for some specific interrelation between the DNA length, the length of the palindromic region and the length of the purine--pyrimidine region. The calculations show that in this case the Z form is formed first with the increasing superhelix density, that the cruciform structure is formed whereas the purine--pyrimidine region returns into the B form, and finally, the Z form is formed again. The possibility of experimental observation of such unusual behaviour is discussed.     

Kinetics of cruciform formation and stability of cruciform structure in superhelical DNA

This is a study of the kinetics of formation of a cruciform structure from the longest palindromic sequence in plasmid pAO3 DNA. DNA was prepared so as to be free of cruciforms even in topoisomers whose negative superhelicity was great enough to induce cruciform formation. Samples of such DNA were incubated at various temperatures, the incubation time varying over a wide range. Then the state was frozen by chilling. Two-dimensional electrophoretic analysis made it possible to estimate the fraction of molecules that got the cruciform structure during incubation. Precautions were taken for electrophoresis conditions to rule out any spontaneous conformational changes within the palindromic region. The relaxation time at the midpoint of the transition ranged from 30 min at 30 C to 50 hrs at 20 C, both in 0.1SSC. An increase in the negative superhelical density by 0.01 led to a 500-fold reduction of the relaxation time at 30 C but had little effect at 20 C. The probability of cruciform formation has been examined as a function of temperature. It has been shown that the cruciform state is no longer the predominant one at elevated temperatures: the cruciformation probability drops to an insignificant value for all of the topoisomers involved. Data have been obtained suggesting that the cruciform formation at the major palindromic site is not the only structural transition possible in pAO3 DNA.     

Kinetics of Cruciform Formation and Stability of Cruciform Structure in Superhelical DNA

Abstract

This is a study of the kinetics of formation of a cruciform structure from the longest palindromic sequence in plasmid pA03 DNA. DNA was prepared so as to be free of cruciforms even in topoisomers whose negative superhelicity was great enough to induce cruciform formation. Samples of such DNA were incubated at various temperatures, the incubation time varying over a wide range. Then the state was frozen by chilling. Two-dimensional electrophoretic analysis made it possible to estimate the fraction of molecules that got the cruciform structure during incubation. Precautions were taken for electrophoresis conditions to rule out any spontaneous conformational changes within the palindromic region. The relaxation time at the midpoint of the transition ranged from 30 min at 30 C to 50 hrs at 20 C, both in 0.1SSC. An increase in the negative superhelical density by 0.01 led to a 500-fold reduction of the relaxation time at 30 C but had little effect at 20 C. The probability of cruciform formation has been examined as a function of temperature. It has been shown that the cruciform state is no longer the predominant one at elevated temperatures: the cruciformation probability drops to an insignificant value for all of the topoisomers involved. Data have been obtained suggesting that the cruciform formation at the major palindromic site is not the only structural transition possible in pA03 DNA.     

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.     

Interaction of a protein from rat liver nuclei with cruciform DNA.

We constructed a synthetic cruciform DNA which closely resembles Holliday junctions, a DNA structure formed during recombination or following the transition from interstrand to intrastrand base pairing in palindromic DNA sequences. We identified and partially purified a protein from rat liver that specifically binds to this cruciform DNA molecule and does not bind to single-stranded or double-stranded DNAs of the same sequence. This protein also binds to the cruciform structure formed by a 70 bp palindromic sequence cloned in plasmid pUC18. No detectable nucleolytic activity is associated with the rat liver cruciform-binding protein, in contrast to all cruciform-recognizing proteins known so far.     

Influence of DNA sequence and supercoiling on the process of cruciform formation

We have studied some of the effects of DNA sequence and negative superhelicity on the rate of cruciform formation. Replacing the sequence AATT at the center of a perfect 68 base-pair palindromic sequence with the sequence CCCGGG decreases the rate of cruciform formation by a factor of at least 100. The logarithm of the rate constant of cruciform formation was found to increase linearly with linking difference. For the 68 base-pair perfect palindrome in a 4400 base-pair plasmid, each additional negative superhelical turn increased the rate of cruciform formation by a factor of 1.6. These results are consistent with a mechanism in which cruciform formation is initiated by the formation of a single-stranded bubble, 10 base-pairs in length, near the center of the palindromic sequence. In addition, we have examined the effect of introducing an asymmetric insertion into the palindromic sequence.     

A 140-Bp-Long Palindromic Sequence Induces Double-Strand Breaks during Meiosis in the Yeast Saccharomyces Cerevisiae

Palindromic sequences have the potential to form hairpin or cruciform structures, which are putative substrates for several nucleases and mismatch repair enzymes. A genetic method was developed to detect such structures in vivo in the yeast Saccharomyces cerevisiae. Using this method we previously showed that short hairpin structures are poorly repaired by the mismatch repair system in S. cerevisiae. We show here that mismatches, when present in the stem of the hairpin structure, are not processed by the repair machinery, suggesting that they are treated differently than those in the interstrand base-paired duplex DNA. A 140-bp-long palindromic sequence, on the contrary, acts as a meiotic recombination hotspot by generating a site for a double-strand break, an initiator of meiotic recombination. We suggest that long palindromic sequences undergo cruciform extrusion more readily than short ones. This cruciform structure then acts as a substrate for structure-specific nucleases resulting in the formation of a double-strand break during meiosis in yeast. In addition, we show that residual repair of the short hairpin structure occurs in an MSH2-independent pathway.     

Large-scale stable opening of supercoiled DNA in response to temperature and supercoiling in (A + T)-rich regions that promote low-salt cruciform extrusion.

We have studied the properties of (A + T)-rich sequences derived from ColE1 that promote cruciform extrusion at low ionic strength in supercoiled plasmids. We compared the chemical reactivity of the sequences in negatively supercoiled DNA (using osmium tetroxide and bromoacetaldehyde) with the results of two-dimensional gel electrophoresis performed under the same conditions. Taken together, the results indicate the occurrence of cooperative helix-coil transitions in the (A + T)-rich DNA at low ionic strength, to form stable, denatured regions. The extent of the open region is a function of temperature and superhelix density, with an additional local destabilization brought about by the presence of cruciform structures. We present a simple statistical mechanical model of the helix-coil transition in the (A + T)-rich DNA, from which we have obtained estimates of the free energy for average base-pair opening of 0.31 kcal mol-1 and that for the formation of a helix-coil junction of 4.9 kcal mol-1, in 45 mM Tris-borate, pH 8.3, 0.5 mM EDTA. The results offer a model for the C-type mechanism of cruciform extrusion. Inverted repeats that are incorporated into the melted region undergo hairpin loop formation below 50 degrees C, and upon closure of the melted region, by reduction of temperature or increased ionic strength, they remain as a fully extruded cruciform structure.     

The interactions of enzyme and chemical probes with inverted repeats in supercoiled DNA

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as S1, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endonucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (delta Gf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high delta Gf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured delta Gf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.     

Flanking AT-rich sequences may lower the activation energy of cruciform extrusion in supercoiled DNA

In the absence of flanking AT-rich segments, cruciform transition energies of DNA palindromic sequences of random base composition are high and mainly dependent upon the base-stacking and -pairing parameters of the palindromic segment. When AT-rich sequences adjoin palindromes, the transition energy of cruciform extrusion is significantly lowered. An inverse relationship exists between the length of the AT-rich stretch and the cruciform transition energy. Long stretches lower the transition energies more than short stretches. At physiological salt and temperature conditions, equilibrium between cruciform extrusion and absorption for the inverted repeat sequences IRS-B and IRS-C of pBR322 derived plasmids is reached in less than five minutes.     

Detection of cruciform extrusion in DNA by temperature-gradient gel electrophoresis

Repetitive sequences in DNA molecules, some of which are palindromic, tend to form stable cruciforms. These are frequently located in promoter regions of a specific operon and origin of replication. Temperature gradient gel electrophoresis can be used to distinguish among various supercoiled DNA topoisomers and to ascertain whether or not the cruciform motif has been extruded. In the current study, this technique is implemented for the first time to address the role of temperature in cruciform extrusion from plasmids.     

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.     

Diethyl pyrocarbonate: a chemical probe for DNA cruciforms.

Two palindromic DNA sequences were analyzed with respect to their chemical reactivities with diethyl pyrocarbonate. In negatively supercoiled plasmid templates enhanced N7 carbethoxylation was found with individual purines located in presumptive single-stranded loops of DNA cruciform structures. No enhanced reactivity at these positions was observed in linear, relaxed or low superhelical density plasmids. Hyperreactivity was found over a narrow region only, indicating that stable cruciforms contain loops of minimal size. No enhanced chemical reactivity was found with the four-way junction at the base of cruciforms. Diethyl pyrocarbonate has proved a sensitive structural probe for the analysis, with single nucleotide resolution, of DNA cruciform structures.     

Localized chemical hyperreactivity in supercoiled DNA: evidence for base unpairing in sequences that induce low-salt cruciform extrusion

Certain A + T-rich DNA sequences (C-type inducing sequences) cause adjacent inverted repeats to undergo cruciform extrusion by a particular pathway (C-type extrusion), which is characterized by large activation energies and extrusion at low salt concentrations and relatively low temperatures. When they are supercoiled, these sequences become reactive toward the normally single-strand-selective reagents bromoacetaldehyde, glyoxal, osmium tetraoxide, and sodium bisulfite. The following evidence is presented: (1) The most reactive sequences are those to the left of the inverted repeat. (2) Chemical reactivity is suppressed by either sodium chloride or micromolar concentrations of distamycin. The suppression of reactivity closely parallels that of C-type cruciform extrusion. (3) Chemical reactivity requires a threshold level of negative supercoiling. The threshold superhelix density depends on the prevailing salt concentration. (4) Analysis of temperature dependences suggests that reaction with osmium tetraoxide involves transient unstacking events, while bromoacetaldehyde requires larger scale helix opening. Thus a variety of opening events may occur in the supercoiled A + T-rich sequences, from small-amplitude breathing to low-frequency, large-amplitude openings. The latter appear to be responsible for C-type cruciform extrusion.     

The Interactions of Enzyme and Chemical Probes with Inverted Repeats in Supercoiled DNA

Abstract

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as SI, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endo- nucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.     

Evidence for allosteric transitions in secondary structure induced by superhelical stress

Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the torsion constants (alpha) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the torsion constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, or vice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae.

We describe a general physical method for detecting the heteroduplex DNA that is formed as an intermediate in meiotic recombination in the yeast Saccharomyces cerevisiae. We use this method to study the kinetic relationship between the formation of heteroduplex DNA and other meiotic events. We show that strains with the rad50, but not the rad52, mutation are defective in heteroduplex formation. We also demonstrate that, although cruciform structures can be formed in vivo as a consequence of heteroduplex formation between DNA strands that contain different palindromic insertions, small palindromic sequences in homoduplex DNA are rarely extruded into the cruciform conformation.