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.     

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.     

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.     

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.     

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.     

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.     

Dynamics of cruciform extrusion in supercoiled DNA: use of a synthetic inverted repeat to study conformational populations.

An inverted repeat has been created in a plasmid by ligation of two 13 nucleotide synthetic oligonucleotides into the cloning vector pAT153. The resulting recombinant plasmid, pIRbke8, is hypersensitive to cleavage by the single-strand-specific nuclease S1, and to modification by the single-strand-selective reagent bromoacetaldehyde, when the plasmid is negatively supercoiled. The new inverted repeat is a stronger S1 site than those derived from pBR322, but, in contrast to the ColE1 and phi X174 RF inverted repeats, these repeats share a similar temperature dependence. The kinetics of EcoRI cleavage at the centre of the synthetic inverted repeat have been studied in supercoiled and linear molecules. It is found that in the supercoiled molecule this target is not refractory to EcoRI cleavage to an extent which is greater than the resolution of the experiment. We conclude that in this molecule the cruciform is in a dynamic equilibrium with the regular duplex, in which the cruciform constitutes a relatively small subpopulation of conformational species.     

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.     

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.     

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.     

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 two-state conformational equilibrium for alternating (A-T)n sequences in negatively supercoiled DNA

We have made a study of the pattern of osmium tetroxide modification in supercoiled plasmids containing alternating (A-T)n tracts. Two distinct alternative patterns may be obtained, depending upon conditions. At moderate salt concentrations, or at low temperature, only thymine bases close to the centres of the tracts were modified, consistent with the presence of a cruciform structure. At higher temperatures in the absence of cations, uniform modification throughout the tracts was observed. The cationic concentration required to stabilize cruciform structure depends markedly on its charge, and a number of transition metal ions were totally ineffective. The results are interpreted in terms of a two-state equilibrium between the cruciform and a perturbed helical structure, the position of which is temperature- and salt-dependent. For longer (A-T)n tracts, a third pattern of osmium tetroxide modification is found at intermediate salt concentrations, consistent with a cruciform having an extensively disrupted four-way junction.     

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.     

The supercoil-stabilised cruciform of ColE1 is hyper-reactive to osmium tetroxide

Supercoiled pColIR215 contains a site of pronounced hyper-reactivity towards modification by osmium tetroxide, a reagent known to be single-strand-selective. The site of hypersensitivity has been mapped to the ColE1 inverted repeat, believed to extrude a cruciform in supercoiled DNA. Linear or relaxed plasmids are not modified by the reagent. We conclude that cruciform formation is responsible for the site-selective modification. Fine mapping of the modification site as a function of time has revealed that the initial reaction occurs at the centre of the inverted repeat, i.e., the unpaired loop of the cruciform, but that the modification region rapidly expands outwards from this point.     

The physical chemistry of cruciform structures in supercoiled DNA molecules

Inverted repeat DNA sequences extrude cruciform structures when present in negatively supercoiled molecules, stabilised by the release of torsional stress brought about by the negative twist change. We have revealed the presence of cruciform structures by means of enzyme and chemical probing experiments and topological band shift methods. The geometry of cruciform structures has been studied from two points of view. The unpairing of bases in the loop region has been investigated using bisulphite modification, with the result that the central four nucleotides have single-stranded character, and the next pair have only partially single-stranded nature. Gel electrophoretic studies of a pseudo-cruciform structure indicate that the cruciform junction introduces a pronounced bend into the molecule. The dependence of the formation of the ColE1 cruciform upon DNA supercoiling shows that it has a free energy of formation of 18.4 +/- 0.5 kcal mole-1. The kinetics of the extrusion process are complex. Most sequences extrude slowly with considerable temperature coefficients, but the detailed properties are strongly sequence-dependent. One synthetic inverted repeat sequence which we have studied in detail has an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole-1. We discuss possible mechanistic pathways for the extrusion process.     

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.     

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.     

Reaction conditions affect the specificity of bromoacetaldehyde as a probe for DNA cruciforms and B-Z junctions.

The reaction of bromoacetaldehyde (BAA) was investigated further with recombinant plasmids containing tracts of (CG)16, in pRW756, or (CA)32, in pRW777, which adopt left-handed Z-structures under the influence of negative supercoiling. The cruciform structures adopted by the inverted repeat sequences near the replication origins of the pBR322 vectors served as internal controls for the number of unpaired bases. The extent of reaction with the B-Z junctions and the cruciforms was dependent on the reaction and analysis conditions, the method of preparation of BAA, ionic conditions, and the amount of negative supercoiling. In contrast to the previous results of Kang and Wells, B-Z junctions in addition to cruciforms do react with BAA. However, more forcing conditions are required to detect this reaction since B-Z junctions appear to be less reactive than the single stranded loops of cruciforms. The site of reaction with DNA was readily mapped with high precision at the nucleotide level. Also, a simple method is described for determining the concentration of BAA as well as its intrinsic reactivity in a given ionic medium.     

Chemical probes of DNA conformation: detection of Z-DNA at nucleotide resolution

Chemical probes sensitive to alterations in DNA conformation, especially Z-DNA, have been identified. These permit cleavage of DNA at sites of unusual structure, the results of which can be displayed on a sequencing gel. Using supercoiled plasmids containing inserts of d(C-G)16 and d(C-A)31 X d(T-G)31, it was found that hydroxylamine and osmium tetraoxide react preferentially with cytosines and thymines, respectively, near B-DNA-Z-DNA junctions; diethylpyrocarbonate reacts more strongly with purines within Z-DNA regions; and dimethylsulfate and diethylsulfate react more strongly with guanines in Z-DNA that are out of phase with the usual pattern of purine-pyrimidine alternation. Our results show that B-Z boundaries are mobile and that with increasing torsional strain, the Z-DNA regions can expand to include nonalternating nucleotide sequences.