Supercoiling-induced DNA bending

Local DNA bending is a critical factor for numerous DNA functions including recognition of DNA by sequence-specific regulatory binding proteins. Negative DNA supercoiling increases both local and global DNA dynamics, and this dynamic flexibility can facilitate the formation of DNA-protein complexes. We have recently shown that apexes of supercoiled DNA molecules are sites that can promote the formation of an alternative DNA structure, a cruciform, suggesting that these positions in supercoiled DNA are under additional stress and perhaps have a distorted DNA geometry. To test this hypothesis, we used atomic force microscopy to directly measure the curvature of apical positions in supercoiled DNA. The measurements were performed for an inherently curved sequence formed by phased A tracts and a region of mixed sequence DNA. For this, we used plasmids in which an inverted repeat and A tract were placed at precise locations relative to each other. Under specific conditions, the inverted repeat formed a cruciform that was used as a marker for the unambiguous identification of the A tract location. When the A tract and cruciform were placed diametrically opposite, this yielded predominantly nonbranched plectonemic molecules with an extruded cruciform and A tract localized in the terminal loops. For both the curved A tract and mixed sequence nonbent DNA, their localization to an apex increased the angle of bending compared to that expected for DNA unconstrained in solution. This is consistent with increased helical distortion at an apical bend.     

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.     

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.     

DNA supercoiling changes the spacing requirement of two lac operators for DNA loop formation with lac repressor.

We have used a gel retardation assay to investigate the influence of DNA supercoiling on loop formation between lac repressor and two lac operators. A series of 15 DNA minicircles of identical size (452 bp) was constructed carrying two lac operators at distances ranging from 153 to 168 bp. Low positive or negative supercoiling (sigma = +/- 0.023) changed the spacing between the two lac operators required for the formation of the most stable loops. This reveals the presence of altered double helical repeats (ranging from 10.3 to 10.7 bp) in supercoiled DNA minicircles. At elevated negative supercoiling (sigma = -0.046) extremely stable loops were formed at all operator distances tested, with a slight spacing periodicity remaining. After relaxation of minicircle-repressor complexes with topoisomerase I one superhelical turn was found to be constrained in those minicircles which carry operators at distances corresponding to a non-integral number of helical turns. This indicates that DNA loop formation can define local DNA domains with altered topological properties of the DNA helix.     

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.     

Structural interconversion of alternating purine-pyrimidine inverted repeats cloned in supercoiled plasmids.

Two self complementary oligonucleotides, T(GC)4AT(GC)4ACATG and C(GC)2(AT)5 (GC)3ATG, were synthesized and cloned into plasmids. Negative supercoiling causes a structural transition in the primary helix of both inserts. The first sequence converts into the left-handed helix, whereas the second sequence undergoes a transition into a cruciform or a Z-type structure depending on the experimental conditions employed. This has been deduced from the mapping of S1 nuclease sensitive sites, OsO4-sensitive sites, DEP modification pattern and relaxation studies. In addition, the differential effect of 5-cytosine methylation and binding of the AT-specific drug distamycin on these transitions further supports this interpretation. Thus, it is demonstrated, that the same sequence which is both inverted repeat and alternating purine-pyrimidine type may adopt either the left-handed conformation or the cruciform structure in response to the superhelical stress. Formation of the Z-type helix can be transmitted through the d(AT)n region which is 10 bp in length.     

(A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable.

Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules. These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease. Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation. These reactivities have a requirement for an alternating sequence. In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound. We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation.     

Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions

An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNA-damaging agent. The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in vitro. The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction. The products of cleavage were unit length linear duplexes that contained terminal hairpin loops. In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 40 bases from an (A-T)34 sequence that extrudes into a cruciform. Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA. The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K. Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca2+, Co2+, or Cu2+. The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration.     

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.