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.     

Tautomeric and microscopic protonation equilibria of some alpha-amino acids

Although there is a wealth of structural and theoretical data relating to palindromic sequences in genomes, the mechanisms of extrusion of cruciform structures during various biological processes in the presence of intercalating agents are still poorly understood. The current study addresses the effects of temperature and intercalator on cruciform extrusion from plasmids and also considers the effects of divalent metal ions on cruciform extrusion. It presents evidence that the cytotoxic effects of certain DNA binding drugs in vivo occur over concentration ranges corresponding to those that modulate cruciform extrusion in vitro. The results confirm earlier studies showing an inverse relationship between the effects of negative superhelicity and temperature on cruciform extrusion. By extrapolation, divalent metal ions facilitate cruciform extrusion by increasing superhelicity. The results allow the concentrations that preclude cruciform extrusion in DNA to be determined, and these are potentially informative about the relationships among temperature, DNA helical winding, cruciform formation, and intercalation. Overall, we provide new and interesting insights into the potential role of cruciform structures in biology and, by implication, cancer therapy.     

Interactions between structure transitions in a torsionally constrained DNA

We used S1 nuclease cleavage in conjunction with gel electrophoresis to evaluate torsion-induced cruciform extrusion at two inverted repeat sequences, IRS-B and IRS-C of plasmid pUC12. These structure transitions affect each other through competition for the available torsional free energy according to their relative energies of activation and the magnitude of DNA duplex unwinding associated with each transition. They can be modulated by the level of DNA negative torsion. Interplays between transition sequences occur over long distances and are independent of relative orientation of transition sites. DNA binding factors that enhance or repress structural transitions of specific sequences may, thus, regulate the structural and functional properties of torsionally coupled, distal sequences.     

Effect of magnesium on cruciform extrusion in supercoiled DNA.

Recently, it was reported that Mg2+greatly facilitates cruciform extrusion in the short palindromes of supercoiled DNA, thereby allowing the formation of cruciform structures in vivo. Because of the potential biological importance of this phenomenon, we undertook a broader study of the effect of Mg2+on a cruciform extrusion in supercoiled DNA. The method of two-dimensional gel electrophoresis was used to detect the cruciform extrusion both in the absence and in the presence of these ions. Our results show that Mg2+shifts the cruciform extrusion in the d(CCC(AT)16GGG) palindrome to a higher, rather than to a lower level of supercoiling. In order to study possible sequence-specific properties of the short palindromes for which the unusual cruciform extrusion in the presence Mg2+was reported, we constructed a plasmid with a longer palindromic region. This region bears the same sequences in the hairpin loops and four-arm junction as the short palindrome, except that the short stems of the hairpins are extended. The extension allowed us to overcome the limitation of our experimental approach which cannot be used for very short palindromes. Our results show that Mg2+also shifts the cruciform extrusion in this palindrome to a higher level of supercoiling. These data suggest that cruciform extrusion in the short palindromes at low supercoiling is highly improbable. We performed a thermodynamic analysis of the effect of Mg2+on cruciform extrusion. The treatment accounted for the effect of Mg2+on both free energy of supercoiling and the free energy of cruciform structure per se. Our analysis showed that although the level of supercoiling required for the cruciform extrusion is not reduced by Mg2+, the ions reduce the free energy of the cruciform structure.     

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.     

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.     

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.     

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.     

Theoretical model of the B-Z transition in DNA with an arbitrary sequence

A statistical-mechanical model is suggested that makes it possible to describe the B-Z transition in DNA with an arbitrary sequence of nucleotides. The key point consists in allowance for the fact that each base pair can assume one of the two states with different energies. One of these states corresponds to the standard Z-form with purines in the syn conformation and pyrimidines in the anti conformation. However, in natural DNA sequences such standard base-pair conformations should be interrupted by energetically unfavorable conformations (syn for pyrimidines and anti for purines). Open regions and cruciform structures are also allowed for in the model. The probabilities of formation of the Z-form stretches, open regions and cruciform structures have been calculated for different values of parameters for pBR322 and pAO3 DNA.     

Chromosomal Translocations Mediated by Palindromic DNA

There is evidence accumulating to suggest that non-B DNA structures have a potential for genomic instability that induces genomic rearrangements including translocations and deletions. One of the best studied examples is the recurrent t(11;22) constitutional translocation in humans that is mediated by palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11. Cloned breakpoint sequences favor adopting a cruciform configuration in vitro. Analysis of the junction fragments implicates frequent double-strand-breaks at the center of both palindromic regions, followed by repair through the non-homologous end joining pathway. De novo examples of the translocation are detected at a substantial frequency in sperm samples from normal healthy males, but not in other normal somatic tissues or cell lines derived from human. Further our recent findings indicate that polymorphism of the PATRR affects the frequency of de novo translocation events and symmetrical alleles preferentially generate the translocation. We propose that the symmetric PATRR is likely to adopt a cruciform structure in male meiotic cells, creating genomic instability that leads to the recurrent translocation.     

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.     

Localization of melted regions in supercoiled DNA by means of chemical modification

Diethyl pyrocarbonate was used as a probe in mapping early melting stages in supercoiled DNA. It was shown that in the process of early melting of pAO3 DNA two denatured regions (about 15 b.p.) arouse near the left and right boundaries of the cruciform structure. In course of further melting denatured regions appeared within AT-rich stretches and the cruciform structure itself disappeared.     

Palindrome-mediated chromosomal translocations in humans

Recently, it has emerged that palindrome-mediated genomic instability contributes to a diverse group of genomic rearrangements including translocations, deletions, and amplifications. One of the best studied examples is the recurrent t(11;22) constitutional translocation in humans that has been well documented to be mediated by palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11. De novo examples of the translocation are detected at a high frequency in sperm samples from normal healthy males, but not in lymphoblasts or fibroblasts. Cloned breakpoint sequences preferentially form a cruciform configuration in vitro. Analysis of the junction fragments implicates frequent double-strand-breaks (DSBs) at the center of both palindromic regions, followed by repair through the non-homologous end joining (NHEJ) pathway. We propose that the PATRR adopts a cruciform structure in male meiotic cells, creating genomic instability that leads to the recurrent translocation.     

The relaxation time for a cruciform structure in superhelical DNA

We have calculated the relaxation time of a cruciform structure in superhelical DNA as a function of the superhelix density for palindromic regions of different lengths. The relaxation time has a sharp maximum at the superhelix density which corresponds to the equilibrium transition point between the cruciform structure and the regular double helix. This maximal value is shown to depend dramatically on the length of the palindromic region.     

Extrusion of an imperfect palindrome to a cruciform in superhelical DNA: complete determination of energetics using a statistical mechanical model

We present a detailed study of the extrusion of an imperfect palindrome, derived from the terminal regions of vaccinia virus DNA and contained in a superhelical plasmid, into a cruciform containing bulged bases. We monitor the course of extrusion by two-dimensional gel electrophoresis experiments as a function of temperature and linking number. We find that extrusion pauses at partially extruded states as negative superhelicity increases. To understand the course of extrusion with changes in linking number, DeltaLk, we present a rigorous semiempirical statistical mechanical analysis that includes complete coupling between DeltaLk, cruciform extrusion, formation of extrahelical bases, and temperature-dependent denaturation. The imperfections in the palindrome are sequentially incorporated into the cruciform arms as hairpin loops, single unpaired bases, and complex local regions containing several unpaired bases. We analyze the results to determine the free energies, enthalpies and entropies of formation of all local structures involved in extrusion. We find that, for each unpaired structure, the DeltaG, DeltaH and DeltaS of formation are all approximately proportional to the number of unpaired bases contained therein. This surprising result holds regardless of the arrangement or composition of unpaired bases within a particular structure. Imperfections have major effects on the overall energetics of cruciform extrusion and on the course of this transition. In particular, the extent of the DeltaLk change necessary to extrude an imperfect palindrome is considerably greater than that required for extrusion of the underlying perfect palindrome. Our analysis also suggests that, at higher temperatures, significant denaturation at the base of an imperfect cruciform can successfully compete with extension of the cruciform arms.     

Nuclease S1-sensitive sites on superhelical DNA molecules carrying the LTR region of Moloney murine leukemia virus

The long terminal repeat (LTR) from proviral DNA of Moloney murine leukemia virus (Mo-MLV) was cloned on a derivative of pBR322, and after introducing superhelical torsions into the resulting recombinant, the sites of conformational transition were investigated by the nuclease S1-digestion method. With an increase in the negative linking differences, fourteen dominant cutting sites were identified, of which two were mapped inside the LTR and one at the 3' end of the LTR. By searching the sequence data, all these sites were localized in the regions having either palindromic sequences or AT-rich sequences. Free energy calculation for the local secondary structure on one strand indicated that nuclease S1 attacked the palindromic sequence regions which could form relatively stable hairpin structures. Under the conditions used, no correlation was found between the S1-sensitive sites and the potential Z-DNA-forming regions, including those within the enhancer sequence.     

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.     

Helix stability and the mechanism of cruciform extrusion in supercoiled DNA molecules

The kinetic properties of cruciform extrusion in supercoiled DNA molecules fall into two main classes. C-type cruciforms extrude in the absence of added salt, at relatively low temperatures, with large activation energies, while S-type cruciforms exhibit no extrusion in the absence of salt, and maximal rates at 50 mM NaCl, with activation energies about one quarter those of the C-type. These diverse properties are believed to reflect two distinct pathways for the extrusion process, and are determined by the nature of the sequences which form the context of the inverted repeat. C-type kinetics are conferred by A + T rich sequences, implying a role of helix stability in the selection. In this study we have shown that: 1. Helix-destabilising solvents (dimethyl formamide and formamide) facilitate extrusion by normally S-type molecules at low temperatures in the absence of salt. 2. C-type extrusion is strongly suppressed by low concentrations (2-4 microM) distamycin, at which concentrations S-type extrusion is enhanced. 3. Some extrusion occurs in a C-type construct in the presence of 50 mM NaCl. This is increased by addition of 3 microM distamycin, under which conditions extrusion becomes effectively S-type. Thus S-type constructs can behave in a quasi-C-type manner in the presence of helix-destabilising solvents, and C-type extrusion is suppressed by binding a compound which stabilises A + T rich regions of DNA. Helix destabilisation leads to C-type behaviour, while helix stabilisation results in S-type properties. These studies demonstrate the influence of contextual helix stability on the selection of kinetic mechanism of cruciform extrusion.     

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.     

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.