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.     

The kinetic properties of cruciform extrusion are determined by DNA base-sequence.

The extrusion kinetics of two cruciforms derived from unrelated DNA sequences differ markedly. Kinetic barriers exist for both reactions, necessitating elevated temperatures before extrusion proceeds at measureable speeds, but the dependence upon temperature and ionic strength is quite different for the two sequences. One, the ColE1 inverted repeat, exhibits a remarkably great temperature dependence of reaction rate and is suppressed by moderate amounts of NaCl or MgCl2. In contrast, the other, a synthetic inverted repeat present in pIRbke8, shows more modest temperature dependence and has a requirement for the presence of salt, with optimal concentrations being 50 mM NaCl or 100 microM MgCl2. Under optimal conditions, cruciform extrusion rates are fast (t1/2 less than 60m) at 37 degrees C for both sequences at native superhelix densities. In 50 mM NaCl the pIRbke8 inverted repeat is characterised by an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole -1. The differences in kinetic properties between the two sequences indicate that DNA base sequence is itself an important factor in determining cruciform kinetics, and possibly even in the selection of the mechanistic 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.     

Evidence for two mechanisms of palindrome-stimulated deletion in Escherichia coli: single-strand annealing and replication slipped mispairing

Spontaneous deletion mutations often occur at short direct repeats that flank inverted repeat sequences. Inverted repeats may initiate genetic rearrangements by formation of hairpin secondary structures that block DNA polymerases or are processed by structure-specific endonucleases. We have investigated the ability of inverted repeat sequences to stimulate deletion of flanking direct repeats in Escherichia coli. Propensity for cruciform extrusion in duplex DNA correlated with stimulation of flanking deletion, which was partially sbcD dependent. We propose two mechanisms for palindrome-stimulated deletion, SbcCD dependent and SbcCD independent. The SbcCD-dependent mechanism is initiated by SbcCD cleavage of cruciforms in duplex DNA followed by RecA-independent single-strand annealing at the flanking direct repeats, generating a deletion. Analysis of deletion endpoints is consistent with this model. We propose that the SbcCD-independent pathway involves replication slipped mispairing, evoked from stalling at hairpin structures formed on the single-stranded lagging-strand template. The skew of SbcCD-independent deletion endpoints with respect to the direction of replication supports this hypothesis. Surprisingly, even in the absence of palindromes, SbcD affected the location of deletion endpoints, suggesting that SbcCD-mediated strand processing may also accompany deletion unassociated with secondary structures.     

Stress-induced cruciform formation in a cloned d(CATG)10 sequence.

The synthetic alternating purine-pyrimidine sequence, d(CATG)10.d(CATG)10, has been cloned into a 2.079-kb pBR322-derived plasmid (pLN1) and its conformation studied under torsional stress. The resultant plasmid, pLNc40, is hypersensitive to cleavage by the single strand-specific nucleases, S1 nuclease and Bal31 nuclease, and to modification by the single strand-selective reagent, osmium tetroxide. The S1-hypersensitive site of this plasmid predominates over those previously mapped in pBR322. Site-specific cleavage of pLNc40 with the resolvase T4 endonuclease VII demonstrates that this alternating purine-pyrimidine tract selectively forms a cruciform structure when stably integrated into a negatively supercoiled plasmid. Quantitative measurements of the twist change (-4.3 +/- 0.2) and free energy of formation (16.2 +/- 0.5 kcal/mol) of this cruciform have been made from two-dimensional gel electrophoresis experiments, and correspond well with the predicted values of cruciform formation for this sequence. We conclude that cruciform extrusion versus the B-Z transition is the favoured conformation of this insert under torsional stress.     

The mechanism of cruciform formation in supercoiled DNA: initial opening of central basepairs in salt-dependent extrusion.

There are two alternative pathways by which inverted repeat sequences in supercoiled DNA molecules may extrude cruciform structures, called C-type and S-type. S-type cruciforms, which form the great majority, are characterised by absolute requirement for cations to promote extrusion, which then proceeds at higher temperatures and with lower activation parameters than for C-type cruciforms. The mechanism proposed for S-type extrusion involves an initial opening of basepairs limited to the centre of the inverted repeat, formation of intra-strand basepairing and a four-way junction, and finally branch migration to the fully extruded cruciform. The model predicts that central sequence changes will be more kinetically significant than those removed from the centre. We have studied the kinetics of cruciform extrusion by a series of inverted repeats related to that of pIRbke8 by either one or two mutations in the symmetric unit. We find that mutations in the central 8 to 10 nucleotides may profoundly affect extrusion rates--the fastest being 2000-fold faster than the slowest, whereas mutations further from the centre affect rates to a much smaller extent, typically up to ten-fold. These data support the proposed mechanism for extrusion via central opening.     

Long range structural communication between sequences in supercoiled DNA. Sequence dependence of contextual influence on cruciform extrusion mechanism

Sequence context may profoundly alter the character of structural transitions in supercoiled DNA (Sullivan, K. M., and Lilley, D. M. J. (1986) Cell 47, 817-827). The A + T-rich sequences of ColE1, which flank the inverted repeat, are responsible for cruciform extrusion following a mechanistic pathway which proceeds via a relatively large denatured region. This C-type mechanism results in kinetic properties which are very different from those of the S-type pathway, the normal mechanism of cruciform extrusion in the absence of the ColE1 flanking sequences. We have analyzed the sequence requirements for the induction of the C-type pathway. The 100-base pair left side sequence of ColE1 (colL) was subjected to systematic deletion using Bal31 exonucleolysis, showing that removal of 30 base pairs from its right end abolished extrusion by the C-type process. A cloned oligonucleotide of the same 30-base pair sequence was sufficient to confer C-type cruciform extrusion on an adjacent inverted repeat. An A + T-rich sequence from Drosophila was found to act like the ColE1 sequences. We have studied the effects of introducing sequences between the A + T-rich colL, and the inverted repeat on which it acts. A range of such fragments was found, from those which augment the effect of colL to those which block it completely. In general, it appears that the ability of a sequence to block the effect of colL depends on both the length and G + C content of the fragment. The sequences which are responsible for the extrusion by the C-type pathway are termed C-type inducing sequences, while sequences which are interposed between the inducing sequence and the inverted repeat, and which may either augment or attenuate the effect, but which cannot function as inducing sequences in isolation, are termed transmitting sequences. The results of these studies are most readily consistent with long range destabilization of DNA structure via telestability effects.     

Long-range structural effects in supercoiled DNA: statistical thermodynamics reveals a correlation between calculated cooperative melting and contextual influence on cruciform extrusion

We have previously described [K. M. Sullivan and D. M. J. Lilley (1986) Cell 47, 817-827] a set of sequences, called C-type inducing sequences, which cause cruciform extrusion by adjacent inverted repeats to occur by an abnormal kinetic pathway involving a large denatured region of DNA. In this paper we apply statistical thermodynamic DNA helix melting theory to these sequences. We find a marked correlation between the ability of sequences to confer C-type cruciform character experimentally and their calculated propensity to undergo cooperative melting, and no exceptions have been found. The correlations are both qualitative and quantitative. Thus the ColE1 flanking sequences behave as single melting units, while the DNA of the S-type plasmid pIRbke8 exhibits no propensity to melt in the region of the bke cruciform. The results of the calculations are also fully consistent with the following experimental observations: 1. the ability of the isolated colL and colR fragments of the ColE1 flanking sequences, as well as the short sequence col30, to confer C-type character; 2. C-type induction by an A + T rich Drosophila sequence; 3. low-temperature cruciform extrusion by an (AT)34 sequence; 4. the effect of changing sequences at a site 90 base pairs (bp) removed from the inverted repeat; 5. the effects of systematic deletion of the colL sequence; and 6. the effects of insertion of various sequences in between the colL sequence and the xke inverted repeat. These studies show that telestability effects on thermal denaturation as predicted from equilibrium helix melting theory of linear DNA molecules may explain all the features that are revealed by studying the extrusion of cruciforms in circular DNA molecules subjected to superhelical stress.     

Interarm interaction of DNA cruciform forming at a short inverted repeat sequence

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.     

Inverted repeat sequences can influence the melting transitions of linear DNAs

The influence of inverted repeat sequences on the melting transitions of linear DNAs has been examined. Derivative melting curves (DMC) of a 514 base pair (bp) DNA, seven subfragments of this DNA, and four other DNAs have been compared to predictions of DNA melting theory. The 514-bp DNA contains three inverted repeat sequences that can form cruciform structures in supercoiled DNA. We refer to these sequences as c-inverted repeats. Previous work showed that the DMC of this DNA, unlike a number of other DNAs, is not accurately predicted by DNA melting theory. Since the theoretical model does not include hairpin-like structures, it was suggested that hairpin or cruciform formation in these inverted repeats may be responsible for this discrepancy. Our results support this hypothesis. Predicted DMCs are in good agreement with DNAs with no inverted repeats, or inverted repeats not evident in supercoiled DNA. Differences between the theoretical and experimental Tm's are less than or equal to 0.3 degrees C. DNA molecules that contain one or more of the three c-inverted repeats are not as accurately predicted. Experimental Tm values are lower than predicted values by 0.7-3.8 degrees C. It is concluded that some inverted repeat sequences can form hairpin-like structures during the melting of linear DNAs. These structures appear to lower overall DNA stability.     

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.     

A dominant influence of flanking sequences on a local structural transition in DNA

We have discovered a striking dependence of a structural transition in DNA on sequences that are distanced from those directly participating in the transformation. The dominant factor determining the selection of kinetic properties of cruciform extrusion is the sequence of the DNA that flanks the inverted repeat. The sequence of the inverted repeat itself appears to have little or no influence. The critical sequences that confer the unusual kinetics exhibited by the ColE1 cruciform are very A+T-rich. A single such sequence is sufficient, which may be as short as 100 bp, and it can control inverted repeats placed at either end. The effects operate in cis, are independent of polarity, and may be effective over relatively long distances. The influence of context has wide implications, possibly including the control of gene expression.     

Thermodynamics of the ColE1 cruciform. Comparisons between probing and topological experiments using single topoisomers

The sensitivity of the ColE1 cruciform to four enzyme and chemical probes of secondary structure has been studied as a function of plasmid topology. Purified topoisomers of pColIR515 have been probed with S1 nuclease, Bal31 nuclease, phage T4 endonuclease VII or osmium tetroxide, and site-specific reaction quantified. Closely similar profiles of reactivity as a function of linking difference were obtained for each probe. Electrophoresis of the pure topoisomers on polyacrylamide/agarose gels revealed a discontinuity in migration as a function of linking difference. Above a threshold linking difference, topoisomers exhibit pronounced reduction in mobility. The linking difference at which this band shift is found correlates precisely with that required for site-specific reaction with the four probes. We conclude that both probing and topological methods are valuable in the study of cruciform structure in supercoiled DNA. The band shift has been measured with accuracy to allow the calculation of the twist change that accompanies the transition, corresponding to delta Tw = -3.2 +/- 0.1. Using this value together with the critical linking difference we calculate a free energy of formation for this structure delta G = 18.4 +/- 0.5 kcal mol-1 (1 kcal = 4.184 kJ).     

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.     

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.     

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.     

Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.     

Effect of base composition at the center of inverted repeated DNA sequences on cruciform transitions in DNA

We have analyzed the effect of base composition at the center of symmetry of inverted repeated DNA sequences on cruciform transitions in supercoiled DNA. For this we have constructed two series of palindromic DNA sequences: one set with differing center and one set with differing center and arm sequences. The F series consists of two 96-base pair perfect inverted repeats which are identical except for the central 10 base pairs which consist of pure AT or GC base pairs. The S series was constructed such that the overall base composition of the inverted repeats was identical but in which the positioning of blocks of AT- and GC-rich sequences varied. The rate of cruciform formation for the inverted repeats in plasmid pUC8 was dramatically influenced by the 8-10 base pairs at the center of the inverted repeat. Inverted repeats with 8-10 AT base pairs in the center were kinetically much more active in cruciform formation than inverted repeats with 8-10 GC base pairs in the center. These experiments show a dominant influence of the center sequences of inverted repeats on the rate of cruciform formation.