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.     

Structural perturbation in supercoiled DNA: hypersensitivity to modification by a single-strand-selective chemical reagent conferred by inverted repeat sequences.

Bromoacetaldehyde, a reagent which modifies unpaired adenine residues, selectively modifies supercoiled DNA in the region of inverted repeats which are known targets for single-strand-specific nucleases. The reaction is dependent upon the topological state of the molecule, and the absolute importance of the inverted repeat has been demonstrated. Finer mapping of the distribution of the modification pattern reveals significant and interesting differences from the S1 nuclease target positions. Bromoacetaldehyde modification is distributed over a wider region covering the whole inverted repeat, with greatest extent of reaction in the regions which flank the inverted repeat. It is suggested that an altered conformation may be propagated into these sequences. These results further support the contention that inverted repeats adopt an altered conformation when negatively supercoiled, for which the principal suggestion remains the cruciform structure.     

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.     

Relationship between superhelical density and cruciform formation in plasmid pVH51

The relative stability of the cruciform state at the large inverted repeat of plasmid pVH51 is measured. At physiological superhelical densities, the cruciform state is present in a high percentage of the plasmid molecules. Investigation of the relationship between negative superhelical density and cruciform prevalence reveals a sharp transition from an undetectable level to a relatively stable state. This transition occurs over the negative superhelical density range of 0.046 to 0.066. Estimates of the free energy contribution to cruciform formation resulting from loss of negative superhelical turns suggest that about 22 kcal/mol are required to generate the cruciform structure at this site in pVH51.     

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.     

Real-time detection of cruciform extrusion by single-molecule DNA nanomanipulation

During cruciform extrusion, a DNA inverted repeat unwinds and forms a four-way junction in which two of the branches consist of hairpin structures obtained by self-pairing of the inverted repeats. Here, we use single-molecule DNA nanomanipulation to monitor in real-time cruciform extrusion and rewinding. This allows us to determine the size of the cruciform to nearly base pair accuracy and its kinetics with second-scale time resolution. We present data obtained with two different inverted repeats, one perfect and one imperfect, and extend single-molecule force spectroscopy to measure the torque dependence of cruciform extrusion and rewinding kinetics. Using mutational analysis and a simple two-state model, we find that in the transition state intermediate only the B-DNA located between the inverted repeats (and corresponding to the unpaired apical loop) is unwound, implying that initial stabilization of the four-way (or Holliday) junction is rate-limiting. We thus find that cruciform extrusion is kinetically regulated by features of the hairpin loop, while rewinding is kinetically regulated by features of the stem. These results provide mechanistic insight into cruciform extrusion and help understand the structural features that determine the relative stability of the cruciform and B-form states.     

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.     

S1 sensitive sites in adenovirus DNA.

S1 nuclease has been used as a probe for regions of DNA secondary structure in supercoiled recombinant plasmids containing adenovirus (Ad) DNA sequences. In the sequences examined two S1 sensitive sites were identified in the left-terminal 16.5% of Ad 12 DNA, one of which aligned approximately with an inverted repeat region in the DNA sequence. In addition an S1 sensitive site was dictated by a potential cruciform structure in the region of the Ad 2 major late promoter. In contrast to the expected cleavage site at the loop of the cruciform, cleavage occurred at the base of the stem in the region of the TATA box. All three S1 sensitive sites identified were more sensitive to S1 than the endogenous sites in the parent plasmids.     

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.     

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.     

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.     

Cruciform extrusion facilitates intramolecular triplex formation between distal oligopurine.oligopyrimidine tracts: long range effects.

Two short d(AG)n tracts separated by either of two 40-base pair (bp) inverted repeats adopt a triple-stranded conformation (H-DNA) at the bottom of an extruded cruciform stem in negatively supercoiled plasmids at pH 4.5. Plasmids containing one d(AG)n adjacent to the inverted repeat or containing two d(AG)n tracts separated by a random sequence did not form the triplex structures. These conclusions were derived from chemical modification patterns and UV-sensitivity studies. Two-dimensional agarose gel electrophoresis revealed that the entire insert containing the cruciform and the triplex is locally unlinked. Moreover, a long range structural effect (over 40 bp of random sequence) of one (AG)7 block on the behavior of a second (AG)7 sequence was detected. This effect cannot be transmitted throughout a 50-bp segment of random sequence. A GenBank search revealed the frequent occurrence of short oligopurine blocks with an intervening random sequence of 17-40 bp.     

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.     

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.     

Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?

pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter.     

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.     

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.     

Target site recognition by a diversity-generating retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

Characterization of an unusual DNA length polymorphism 5'' to the human antithrombin III gene

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.