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.     

Gel electrophoresis of partially denatured DNA. Retardation effect: its analysis and application.

The hypothesis about the role of partial denaturation in DNA retardation during its electrophoresis in denaturing gel /1,2/ was tested. We used partially melted DNA molecules in which the size of the melted regions and their location were known. They were obtained through glyoxal treatment of the melted regions by a procedure allowing the denatured state to be fixed at any point within the melting range. The approach and the availability of the melting maps of DNAs made it possible to investigate DNA molecules differing in length and in the size of the melted regions. The presence of a denatured region at the end of the molecule or inside of it was shown to decrease its electrophoretic mobility, the effect depending on the size of the melted region and on the DNA length. On the basis of the experimental results an explanation is proposed for the cause of retardation in the case of partially denatured DNA.     

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.     

DNA of the Streptomyces phage SH10: Binding sites for Escherichia coli RNA polymerase and denaturation map

Summary Escherichia coli RNA polymerase bound to Streptomyces phage SH10 DNA was visualized by electron microscopy. Six specific binding sites were observed at map units 53, 85, 93, 97, 98, and 99 on the physical map of the 48 kb long genome. Electron microscopy of partially denatured SH10 DNA revealed a characteristic melting pattern of A+T-rich regions around map units 1, 3, 48, 52, and 99. A comparison of the denaturation map with the RNA polymerase binding sites indicates that three binding sites are located in the most A+T-rich regions, two in other early melting regions and one in a segment of higher DNA helix stability.     

A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.     

Electron microscope maps of SA7 DNA melting

Electron microscopic denaturation maps corresponding to the first peaks of the differential melting curve of SA7 DNA were constructed by fixation of partly denatured molecules with glyoxal at temperatures within the melting range. These maps were oriented with respect to the functional map of the virus genome. The localization and the size of the most AT-rich SA7 DNA regions were determined.     

Immunochemical studies on the correlation between conformational changes of DNA caused by ultraviolet irradiation and manifestation of antigenicity.

The conformational changes of double-stranded DNA induced during irradiation with ultraviolet, were immunologically investigated. These studies revealed that at least two distinct antigenic sites were induced in the irradiated DNA molecule, giving rise to two different antibodies specific for ultraviolet-irradiated (uv) DNA and thermally denatured DNA-like structure, and these were demonstrable using radioimmunoassay and double diffusion tests. A series of experiments, including melting profile, fluorescence intensity of the ethidium bromide complex and chromatographic behavior on hydroxyapatite, performed on the antigenically active uvDNA indicated that the duplex structure of DNA separated irregularly during irradiation. Furthermore, the data showed that the conformational determinants of uvDNA are located on the exposed single-stranded regions.     

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.     

The relaxation time for a cruciform structure in superhelical DNA

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

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.     

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.     

Calculation of melting curves for DNA

A method is reported for calculating the melting curve of a DNA molecule of random base sequence, including in the formalism the dependence of the free energy of base pair formation on the size of a denatured section. Some explicit results are shown for a “typical” base sequence, in particular the probability of helix formation at individual base pairs in several different regions of the molecule and the amount of melting from the end of the chain. Particular attention is drawn to the variation of local melting behavior from one region of the molecule to another. It is found that sections rich in AT melt at relatively low temperatures with a fairly broad transition curve, whereas regions rich in GC pairs melt at higher temperatures (as expected) with a very abrupt, local transition curve. To account qualitatively for the results one may divide melting into two kinds of processes: (a) the nucleation and growth of denatured regions, and (b) the merging together of two denatured sections at the expense of the intervening helix. The first of these processes dominates in the first stages of melting, and leads to rather broad local melting curves, whereas the second process predominates in the later stages, and occurs, in a particular part of the molecule, over a very narrow temperature range. It is estimated that the average length of a helix plus adjacent coil section at the midpoint of the transition is approximately 600 base pairs. Since transition curves which measure the local melting behavior reflect local compositions fluctuations, these curves contain information about the broad outlines of base sequence in the molecule. Some suggestions are made concerning experiments by which this potential information source could be exploited. In particular, it is pointed out that one might hope to map AT or GC rich regions at particular genetic loci in a biologically active DNA molecule. Values of the relevant parameters found earlier for the transition of homopolymers produce melting curves for a DNA of random base sequence which are in good agreement with the experimental transition curve for T2 phage DNA. Hence the present theoretical picture of the melting of polynucleotides is at least internally self-consistent.     

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.     

Sequence-specific functions of the early palindrome domain within the SV40 core origin of replication.

The early palindrome domain within the SV40 core origin of replication is essential for the initiation of replication. Studies with single point mutants in this region suggested that the early palindrome domain does not function as a cruciform structure, but may be involved in the initiation of SV40 DNA replication in a sequence-specific manner. Two mutants, base-substituted at a primase initiation site nucleotide 5214, showed dramatic decreases in DNA replication in monkey cells. Despite earlier reports to the contrary, disruption of the cruciform configuration or polypyrimidine tract does not invariably lead to lack of replication function, as some mutants unable to form this structure replicate normally. Gel retention assays and DNase I footprinting with the nuclear proteins of monkey cells showed that the 5'GAGGC3' pentanucleotide repeats on either side of early palindrome domain interact with monkey nuclear protein. The early palindrome domain may affect the interaction of SV40 DNA with nuclear protein, and participate in SV40 DNA replication.     

Why is the denaturation process of super-helical DNA so uncooperative?

A theoretical model is proposed for the helix-coil transition in circular covalently closed DNA. In the melting interval the topological constrains for such a molecule are considered to affect both the inner state of the melted regions and the spatial configuration of the whole molecule ("writhing"). A good agreement is achieved between the theoretically calculated and experimentally obtained parameters of the denaturation process. A conclusion is drawn, that at the early stages of thermodenaturation (approximately till the transition midpoint) the effect of topological constrains is realised mainly through the writhing of the molecule. The denatured regions exist in the form of relaxed loops.     

Visualization of the cruciform structure of superhelical DNA by use of atomic force microscopy

Atomic force microscopy was used to visualize the cruciform structure in supercoiled plasmid pUC8 DNA immobilized on aminomodified mica. The cruciform hairpin was 14 base pairs in size, as determined from atomic force microscopy images of pUC8 DNA in air. Molecular modeling confirmed that the cruciform structure is formed by hairpins with self-complementary homopyrimidine-homopurine sequences (dT)8(dA)6 and a loop 4 nucleotides long.     

Polarographic reducibility of denatured DNA

The de polarographic behavior of native and denatured DNA at pH 7.0 was studied. Whereas native DNA was polarographically inactive under the given conditions, denatured DNA yielded a reduction polarographic step at the potential of about ?1.4 V. Native DNA produced a single desorption wave on ac polarograms, while denatured DNA yielded, in addition to this wave, another more negative wave approximately corresponding, as to its potential, to the dc polarographic step of denatured DNA. The behavior of apurinic acid was similar to that of denatured DNA. The course of DNA denaturation at elevated temperature was studied by means of the two above techniques and changes at temperatures below the melting temperature observed. This finding is in agreement with earlier results obtained by oscillopolarographic and the pulse-polarographic method.     

Physical studies of chloroacetaldehyde labelled fluorescent DNA

The reaction of chloroacetaldehyde with denatured DNA produces a fluorescent DNA where both the adenine and cytosine bases are modified. The rate of modification of DNA by chloroacetaldehyde was measured using the absorption spectrum shift. The depolarization and quantum yield of native DNA and denatured DNA were investigated as a function of temperature.The melting points and the renaturation rates of a series of derivative DNA's were investigated. The melting point was decreased by 1.3°C for each base modified per 100 base pairs corresponding to a 2.8 Kcal destabilizing free energy per mismatched base pair. The renaturation rate of the derivative DNA is reduced by a factor 2 when the melting temperature is lowered by 13°C.