DNA supercoiling enables the type IIS restriction enzyme BspMI to recognise the relative orientation of two DNA sequences

Many proteins can sense the relative orientations of two sequences at distant locations in DNA: some require sites in inverted (head-to-head) orientation, others in repeat (head-to-tail) orientation. Like many restriction enzymes, the BspMI endonuclease binds two copies of its target site before cleaving DNA. Its target is an asymmetric sequence so two sites in repeat orientation differ from sites in inverted orientation. When tested against supercoiled plasmids with two sites 700 bp apart in either repeated or inverted orientations, BspMI had a higher affinity for the plasmid with repeated sites than the plasmid with inverted sites. In contrast, on linear DNA or on supercoiled DNA with sites 1605 bp apart, BspMI interacted equally with repeated or inverted sites. The ability of BspMI to detect the relative orientation of two DNA sequences thus depends on both the topology and the length of the intervening DNA. Supercoiling may restrain the juxtaposition of sites 700 bp apart to a particular alignment across the superhelical axis, but the juxtaposition of sites in linear DNA or far apart in supercoiled DNA may occur without restraint. BspMI can therefore act as a sensor of the conformational dynamics of supercoiled DNA.     

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.     

Fluctuations in superhelical DNA.

The effect of superhelicity on the base-pair opening probability and on the probability of occurrence of cruciform states in palindromic regions is theoretically treated. The calculations show that below the superhelix density value of -sigma=0.05 superhelicity does not appreciably affect the characteristics of DNA secondary structure fluctuations. In the range of physiological superhelix densities sigma (-sigma=0.05-0.09) the base-pair opening probability markedly increases. However, within this range of sigma the base-pairs are opened only transiently and permanently open regions are not formed. Permanently opened regions appear at higher negative superhelix densities (-sigma greater than 0.10). At the values of -sigma higher than 0.06 a cruciform structure in the palindromic region centred in position 3965 proves to be the most probable fluctuational disturbance in the 0x174 duplex DNA. Different experimental approaches used for probing the fluctuations in superhelical DNA have been analysed. The results suggest that most direct quantitative information can be derived from data on the nicking of closed DNA by single strand-specific endonucleases. Such data (Wang, 1974) accord with the results of theoretical calculations. Calculations show that, due to base-pair opening, the total free energy of superhelical DNA should depend parabolically on sigma only up to some critical value of sigma=sigmac. If negative superhelicity exceeds this critical value, which under physiological conditions proves to be -sigma=0.085, the free energy should increase linearly with -sigma. The biological role of supercoiling is discussed in the light of obtained results.     

The dihedral symmetry of the p53 tetramerization domain mandates a conformational switch upon DNA binding.

The p53 tumor suppressor forms stable tetramers, whose DNA binding activity is allosterically regulated. The tetramerization domain is contained within the C-terminus (residues 323-355) and its three-dimensional structure exhibits dihedral symmetry, such that a p53 tetramer can be considered a dimer of dimers. Under conditions where monomeric p53 fails to bind DNA, we studied the effects of p53 C-terminal mutations on DNA binding. Residues 322-355 were sufficient to drive DNA binding of p53 as a tetramer. Within this region residues predicted by the three-dimensional structure to stabilize tetramerization, such as Arg337 and Phe341, were critical for DNA binding. Furthermore, substitution of Leu344 caused p53 to dissociate into DNA binding-competent dimers, consistent with the location of this residue at the dimer-dimer interface. The p53 DNA site contains two inverted repeats juxtaposed to a second pair of inverted repeats. Thus, the four repeats exhibit cyclic-translation symmetry and cannot be recognized simultaneously by four dihedrally symmetric p53 DNA binding domains. The discrepancy may be resolved by flexible linkers between the p53 DNA binding and tetramerization domains. When these linkers were deleted p53 exhibited novel DNA binding properties consistent with an inability to recognize four contiguous DNA repeats. Allosteric regulation of p53 DNA binding may involve repositioning the DNA binding domains from a dihedrally symmetric state to a DNA-bound asymmetric state.     

Searching for target sequences by p53 protein is influenced by DNA length

One of the most important functions of the tumor suppressor p53 protein is its sequence-specific binding to DNA. Using a competition assay on agarose gels we found that the p53 consensus sequences in longer DNA fragments are better targets than the same sequences in shorter DNAs. Semi-quantitative evaluation of the competition experiments showed a correlation between the relative p53-DNA binding and the DNA lengths. Our results are consistent with a model of the p53-DNA interactions involving one-dimensional migration of the p53 protein along the DNA for distances of about 1000 bp while searching for its target sites. Positioning of the p53 target in the DNA fragment did not substantially affect the apparent p53-DNA binding, suggesting that p53 can slide along the DNA in a bi-directional manner. In contrast to full-length p53, the isolated core domain did not show any significant correlation between sequence-specific DNA binding and fragment length.     

A DNA conformational alteration induced by a neighboring oligopurine tract on GAATTA enables nicking by EcoRI

The pseudo EcoRI site GAATTA in the U3 region of the long terminal repeat of human immunodeficiency virus, which is flanked by a 26-base pair oligopurine tract, is readily nicked by either EcoRI or RsrI. The strand-specific nick occurs predominantly between the G and A residues and is independent of negative supercoiling. Other GAATTA sites surrounded by random (non-oligopurine) sequences are not nicked by these restriction endonucleases. However, other types and lengths of oligopurine tracts are effective in inducing the nicking in neighboring GAATTA sites. Hence, we propose that the flanking oligopurine tracts induce an altered DNA conformation on the GAATTA target site which may be similar to the transition state induced by EcoRI when binding to its canonical recognition site. Gel retardation analyses on restriction fragments containing the oligopurine-GAATTA-oligopurine sequences suggest the presence of helical axis distortions which are consistent with this interpretation.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

Counterintuitive DNA Sequence Dependence in Supercoiling-Induced DNA Melting

The metabolism of DNA in cells relies on the balance between hybridized double-stranded DNA (dsDNA) and local de-hybridized regions of ssDNA that provide access to binding proteins. Traditional melting experiments, in which short pieces of dsDNA are heated up until the point of melting into ssDNA, have determined that AT-rich sequences have a lower binding energy than GC-rich sequences. In cells, however, the double-stranded backbone of DNA is destabilized by negative supercoiling, and not by temperature. To investigate what the effect of GC content is on DNA melting induced by negative supercoiling, we studied DNA molecules with a GC content ranging from 38% to 77%, using single-molecule magnetic tweezer measurements in which the length of a single DNA molecule is measured as a function of applied stretching force and supercoiling density. At low force (<0.5pN), supercoiling results into twisting of the dsDNA backbone and loop formation (plectonemes), without inducing any DNA melting. This process was not influenced by the DNA sequence. When negative supercoiling is introduced at increasing force, local melting of DNA is introduced. We measured for the different DNA molecules a characteristic force F _char, at which negative supercoiling induces local melting of the dsDNA. Surprisingly, GC-rich sequences melt at lower forces than AT-rich sequences: F _char = 0.56pN for 77% GC but 0.73pN for 38% GC. An explanation for this counterintuitive effect is provided by the realization that supercoiling densities of a few percent only induce melting of a few percent of the base pairs. As a consequence, denaturation bubbles occur in local AT-rich regions and the sequence-dependent effect arises from an increased DNA bending/torsional energy associated with the plectonemes. This new insight indicates that an increased GC-content adjacent to AT-rich DNA regions will enhance local opening of the double-stranded DNA helix.     

Topological effects of the TATA box binding protein on minicircle DNA and a possible thermodynamic linkage to chromatin remodeling

DNA ring closure experiments on short restriction fragments ( approximately 160 bp) bound by the TATA box binding protein (TBP) have demonstrated the formation of negative topoisomers, consistent with crystallographically observed TBP-induced DNA untwisting but in contrast to most previous results on topological effects in plasmid DNA. The difference may be due to the high free energy cost of substantial writhe in minicircles. A speculative mechanism for the loss of TBP-induced writhe suggests that TBP is capable of inducing DeltaTw between 0 and -0.3 in minicircles, via loss of out-of-plane bending upon retraction of intercalating Phe stirrups, and that TBP can thus act as a "supercoil shock absorber". The proposed biological relevance of these observations is that they may model the behavior of DNA in constrained chromatin environments. Irrespective of the detailed mechanism of TBP-induced supercoiling, its existence suggests that chromatin remodeling and enhanced TBP binding are thermodynamically linked. Remodeling ATPases or histone acetylases release some of the negative supercoiling previously restrained by the nucleosome. When TBP takes up the supercoiling, its binding should be enhanced transiently until the unrestrained supercoiling is removed by diffusion or topoisomerases. The effect is predicted to be independent of local remodeling-induced changes in TATA box accessibility.     

Histone H1 preferentially binds to superhelical DNA molecules of higher compaction.

In chromatin, the physiological amount of H1 is one molecule per nucleosome or, roughly, one molecule per 200 bp of DNA. We observed that at such a stoichiometry, H1 selectively binds to supercoiled DNA with magnitude of sigma > or = 0.012 (both negative and positive), leaving relaxed, linear, or nicked DNA molecules unbound. When negative and positive DNA topoisomers of varying superhelicity are simultaneously present in the binding mixture, H1 selectively binds to the molecules with highest superhelicity; less supercoiled forms are gradually involved in binding upon increasing the amount of input protein. We explain this topological preference of H1 as the consequence of an increased probability for more than one H1-DNA contact provided by the supercoiling. The existence of simultaneous contacts of H1 with both intertwined DNA strands in the supercoiled DNA molecules is also inferred by topoisomerase relaxation of H1-DNA complexes that had been prefixed with glutaraldehyde.     

The role of negative supercoiling in Hin-mediated site-specific recombination.

A series of biochemical assays were developed and performed to monitor the molecular events that occur during the Hin-mediated DNA inversion reaction. These events can be divided into five different stages: 1) binding of proteins (Hin, Fis, and HU) to DNA; 2) pairing of Hin-binding sites; 3) invertasome formation; 4) DNA strand cleavage; 5) strand rotation and religation. A series of topoisomers of the wild type DNA substrate plasmid (ranging from fully relaxed molecules to those with more than the physiological superhelical density (the physiological superhelical density of pKH336 from Escherichia coli DH10B is -0.072 in this study)) was generated, and the role of negative supercoiling in each step of the inversion reaction was investigated. We found differences in the dependence of the formation of paired Hin-binding sites and of the invertasome formation on the superhelical density of the substrate plasmid. Pairing of Hin-binding sites occurs independently from invertasome formation, and a relatively low degree of negative supercoiling is enough to promote maximal pairing. However, efficient invertasome formation requires higher levels of negative supercoiling.