Highly selective chemical modification of cruciform loops by diethyl pyrocarbonate.

Diethyl pyrocarbonate reacts with the single-stranded loops of cruciform structures with great selectivity. Adenine bases are carbethoxylated, as a result of which the backbone may be cleaved with piperidine, and the level of chemical modification at each base may be determined. We have studied the ColE1 and (A-T)34 cruciforms of pColIR315 and pXG540. In each case we observe maximal modification at the most central adenosine of the loop, and an overall pattern of modification corresponding to a total loop size of about six bases. The results may be interpreted in terms of a model in which the loop has a defined tertiary structure. No modification was detected at either cruciform four-way junction, suggesting that this region is fully base-paired.     

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.     

The dynamic distribution and quantification of DNA cruciforms in eukaryotic nuclei

Cruciforms have been suggested as potential recognition structures at or near origins of DNA replication in eukaryotic cells. Monoclonal antibodies specific for cruciforms have been produced. The antibody binds to structural determinants at the base of the cruciform stem, the "elbow." Labeling of nuclei with anti-cruciform antibodies produces a nonuniform pattern of fluorescence in cells arrested at the G1/S boundary. This pattern of fluorescence changes when these cells are released from synchrony. Using fluorescence flow cytometry to quantify the number of DNA cruciform structures in cells throughout the cell cycle, we observed two major populations of nuclei with different numbers of cruciforms; the modal number of cruciforms in these populations was 0.6 x 10(5) and 3 x 10(5) cruciforms per nucleus. Synchronized cells (doubly arrested by serum starvation and aphidicolin) displayed a biphasic distribution of the number of cruciforms over the first 6 h after release from synchrony with maxima at 0 and 4 h after release.     

Inverted repeats,stem-loops,and cruciforms: Significance for initiation of DNA replication

Inverted repeats occur nonrandomly in the DNA of most organisms. Stem-loops and cruciforms can form from inverted repeats. Such structures have been detected in pro- and eukaryotes. They may affect the supercoiling degree of the DNA, the positioning of nucleosomes, the formation of other secondary structures of DNA, or directly interact with proteins. Inverted repeats, stem-loops, and cruciforms are present at the replication origins of phage, plasmids, mitochondria, eukaryotic viruses, and mammalian cells. Experiments with anti-cruciform antibodies suggest that formation and stabilization of cruciforms at particular mammalian origins may be associated with initiation of DNA replication. Many proteins have been shown to interact with cruciforms, recognizing features like DNA crossovers, four-way junctions, and curved/bent DNA of specific angles. A human cruciform binding protein (CBP) displays a novel type of interaction with cruciforms and may be linked to initiation of DNA replication. © 1996 Wiley-Liss, Inc.     

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.     

The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite.

The single-stranded character of cytosine bases in three cruciform structures has been assessed by an examination of reactivity towards sodium bisulphite. Unpaired cytosine residues undergo deamination at C4 to give deoxyuracil, and propagation in an ung Escherichia coli host results in C-G----T-A transition mutations, detectable by restriction cleavage or sequence analysis. Very high frequencies of such mutations have been found at cruciform loops, confirming their unpaired character, with almost zero background mutation frequencies elsewhere. A low level of modification was observed at the four-way junction of a cruciform. The results indicate that the optimal cruciform loop size is four bases, with loose 'breathing' at the first base pair at the top of the cruciform stem at 37 degrees C, and little or no opening of base pairs at the four-way junction.     

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.     

Rh(DIP)3(3+): a shape-selective metal complex which targets cruciforms.

The coordination complex tris(4,7-diphenylphenanthroline)rhodium(III), Rh(DIP)3(3+), binds to and, upon photoactivation, cleaves both DNA strands near the base of a DNA cruciform. Sites of photoinduced double-stranded DNA cleavage by the rhodium complex map to regions containing cruciforms on closed circular pBR322, pColE1 and phi X174 (replicative form) DNAs. Neither cleavage nor binding by the metal complex, assayed using S1 nuclease, is found on the linear plasmid which lacks the extruded cruciform. High resolution mapping experiments reveal that Rh(DIP)3(3+) cleaves at a specific AT-rich site neighboring the stem of the minor cruciform on pBR322. The primary site of cleavage is found at position 3238 on the 3'-strand and 3250 on the 5'-strand and is remarkably specific. The pattern of cleavage, to one side only of the cruciform stem, indicates an asymmetry in the cruciform structure recognized by the complex. These results suggest that Rh(DIP)3(3+) may provide a useful reagent to probe cruciform sites. In addition, the high degree of specificity found in targeting the cruciform structure with this simple metal complex underscores the utility of shape-selection for the recognition of specific sites on a DNA strand.     

Intermediate range effects in DNA. I: Low pH/stress induced conformational changes in the vicinity of an extruded d(AT)n.d(AT) in cruciform.

A family of plasmids which contain d(AT)n cruciforms are sensitive to "single strand specific" (SS) endonucleases and a variety of chemical probes in a "random sequence" region centered 10-30 residues away from the cruciform junction. The SS nuclease sensitive structures are dependent on the presence of the extruded cruciform and exhibit a degree of sequence independence. Their appearance depends upon the combined effects of slightly lower than neutral pH and superhelical coiling above the minimum required to drive the extrusion of the d(AT)n cruciform arms. The nuclease sensitive structure is therefore underwound with respect to the B conformation and contains protonated bases.     

Occurrence of potential cruciform and H-DNA forming sequences in genomic DNA.

We have used computer-assisted methods to search large amounts of the human, yeast and Escherichia coli genomes for inverted repeat (IR) and mirror repeat (MR) DNA sequence patterns. In highly supercoiled DNA some IRs can form cruciforms, while some MRs can form intramolecular triplexes, or H-DNA. We find that total IR and MR sequences are highly enriched in both eukaryotic genomes. In E. coli, however, only total IRs are enriched, while total MRs only occur as frequently as in random sequence DNA. We then used a set of experimentally derived criteria to predict which of the total IRs and MRs are most likely to form cruciforms or H-DNA in supercoiled DNA. We show that strong cruciform forming sequences occur at a relatively high frequency in yeast (1/19 700 bp) and humans (1/41 800 bp), but that H-DNA forming sequences are abundant only in humans (1/49 400 bp). Strong cruciform and H-DNA forming sequences are not abundant in the E.coli genome. These results suggest that cruciforms and H-DNA may have a functional role in eukaryotes, but probably not prokaryotes.     

A novel type of interaction between cruciform DNA and a cruciform binding protein from HeLa cells.

We recently identified and enriched a protein (CBP) from HeLa cells with binding specificity for cruciform-containing DNA. We have now studied the interaction of CBP with stable cruciform DNA molecules containing the 27 bp palindrome of SV40 on one strand and an unrelated 26 bp palindrome on the other strand by hydroxyl radical footprinting. The CBP-DNA interaction is localized to the four-way junction at the base of the cruciforms. CBP appears to interact with the elbows of the junctions in an asymmetric fashion. Upon CBP binding, structural distortions were observed in the cruciform stems and in a DNA region adjacent to the junction. These features distinguish CBP from other cruciform binding proteins, which bind symmetrically and display exclusively either contacts with the DNA backbone or structural alterations in the DNA.     

Formation of (dA-dT)n cruciforms in Escherichia coli cells under different environmental conditions.

We have detected cruciform formation of (dA-dT)n inserts in Escherichia coli cells by analyzing the superhelical density of isolated plasmid DNA samples and by probing intracellular DNA with chloroacetaldehyde. The plasmids we used were pUC19 containing inserts of (dA-dT)n. The cruciforms appeared after cells underwent different stresses: inhibition of protein synthesis, anaerbiosis, and osmotic shock. At the same time, all these stimuli led to an increase in superhelical density of the control pUC19 plasmid DNA. Therefore, we suggest that the increase in plasmid superhelicity in response to different environmental stimuli entails the appearance of cruciform structures. The use of the (dA-dT)n units of various lengths made it possible to estimate the superhelical density of the plasmid DNA in vivo.     

A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease

T7 endonuclease preferentially cleaves purified supercoiled pBR322 and colE1 plasmids at the single-stranded regions exposed when palindromic sequences assume cruciform structures (Panayotatos, N., and Wells, R.D. (1981) Nature 289, 466-470). In vivo, however, induction of nuclease synthesis off a cloned gene caused complete degradation of the bacterial DNA but not of the plasmid vector; presumably, single-stranded regions (cruciforms?) on the genome effectively complete for the nuclease with similar sites on the plasmid (Panayotatos, N., and Fontaine, A. (1985) J. Biol. Chem. 260, 3173-3177). To overcome this competition, we introduced on the plasmid the naturally occurring colE1 palindrome which forms a more stable cruciform in vitro. In addition, we increased the target size (and the T7 endonuclease gene dosage) by raising the copy number of the plasmid 5-fold. Induction of the endonuclease encoded by this new plasmid (pLAT75) resulted not only in degradation of genomic DNA but also in intracellular nicking and linearization of the plasmid. The cleavage site in vivo was mapped at the colE1 palindrome and coincided with the site cleaved specifically in vitro by either T7 or S1 endonuclease only when this palindrome assumes the cruciform structure. These results indicate that cruciform structures exist intracellularly and demonstrate the usefulness of endonucleases as probes of DNA topology in vivo.     

Length-dependent cruciform extrusion in d(GTAC)n sequences

pBR322-derived plasmids have been constructed carrying d(GTAC)n.d(GTAC)n inserts of different lengths, in order to investigate the effect of insert size on cruciform extrusion and/or the B-Z transition. Plasmids with n ranging from 4 to 12 are hypersensitive to cleavage by the single-strand specific nucleases, S1 nuclease and Bal31 nuclease. Hypersensitive sites associated with the smaller alternating purine-pyrimidine tracts, however, coexist with the major pBR322 sites. Site-selective cleavage of these plasmids with the resolvase, T7 endonuclease I, demonstrates that all the inserts form cruciform structures when stably integrated into negatively supercoiled plasmids. An increase in the negative superhelical density of the DNA's induces cruciform formation within the insert region, resulting in a reduction in torsional stress consistent with the size of the insert. Moreover, as n decreases, the superhelical density required to stabilise the cruciform state increases. Therefore, the cruciform geometry is the favoured conformation of these d(GTAC)n.d(GTAC)n sequences under torsional stress. The stability of these cruciforms increases as n increases, with cruciformation occurring at lower superhelical densities and to the exclusion of the other pBR322 cruciforms.     

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.