Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.     

Structure of plectonemically supercoiled DNA

Using electron microscopy and topological methods, we have deduced an average structure for negatively supercoiled circular DNA in solution. Our data suggest that DNA has a branched plectonemic (interwound) form over the range of supercoiling tested. The length of the superhelix axis is constant at 41% of the DNA length, whereas the superhelix radius decreases essentially hyperbolically as supercoiling increases. The number of supercoils is 89% of the linking deficit. Both writhe and twist change with supercoiling, but the ratio of the change in writhe to the change in twist is fixed at 2.6:1. The extent of branching of the superhelix axis is proportional to the length of the plasmid, but is insensitive to superhelix density. The relationship between DNA flexibility constants for twisting and bending calculated using our structural data is similar to that deduced from previous studies. The extended thin form of plectonemically supercoiled DNA offers little compaction for cellular packaging, but promotes interaction between cis-acting sequence elements that may be distant in primary structure. We discuss additional biological implications of our structural data.     

Compaction of single molecules of supercoiled DNA immobilized on amino mica: from duplex to minitoroidal and spheroidal conformations

Supercoiled DNA pGEMEX with a length of 3993 nucleotides was immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica) and visualized by atomic force microscopy. Plectonemically supercoiled DNA molecules and molecules with an extremely high level of compaction were visualized on modified amino mica, which was characterized by increased surface charge density. It was found that the length of the superhelix axis decreases two and four times to form superhelix axes of the second and third orders as the DNA compaction level increases because of the twice folding of DNA molecules. In this case, the length of the superhelix axis decreases from L approximately 470 nm to L approximately 140 nm (which corresponds to 10% contour length of a relaxed molecule on assumption of B-DNA) to form minitoroids and spheroids of approximately 50 nm diameter. Note that the previously reported experimentally measured length of the superhelix axis was equal to 35% contour length of the relaxed DNA molecule at the maximal density of the superhelix. Our data show that the significant decrease in the length of superhelix axis and the compaction of single supercoiled DNA molecules to the level of spheroids and minitoroids are caused by the screening of negatively charged DNA phosphate groups by positively charged amino groups of the modified amino mica because of its high surface charge density and increased hydrophobicity compared with standard amino mica.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

Intrachain reactions of supercoiled DNA simulated by Brownian dynamics

We considered an irreversible biochemical intrachain reaction of supercoiled DNA as a random event that occurs, with certain probability, at the instant of collision between two reactive groups bound to distant DNA sites. Using the Brownian dynamics technique, we modeled this process for a supercoiled DNA molecule of 2.5 kb length in dilute aqueous solution at an NaCl concentration of 0.1 M. We calculated the mean reaction time tau(Sigma) as a function of the intrinsic second-order rate constant k(I), the reaction radius R, and the contour separation S of the reactive groups. At the diffusion-controlled limit (k(I) --> infinity), the kinetics of reaction are determined by the mean time tau(F) of the first collision. The dependence of tau(F) on R is close to inversely proportional, implying that the main contribution to the productive collisions is made by bending of the superhelix axis. At sufficiently small k(I), the mean reaction time can be satisfactory approximated by tau(Sigma) = tau(F)(app) + 1/(k(I)c(L)), where c(L) is the local concentration of one reactive group around the other, and tau is an adjustable parameter, which we called the apparent time of the first collision. The value of tau depends on R very weakly and is approximately equal to the mean time of the first collision caused by mutual reptation of two DNA strands forming the superhelix. The quasi-one-dimensional reptation process provides the majority of productive collisions at small k(I) values.     

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.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

Formation of noncanonical structures in superhelical DNA. Mutual effects of various transitions

This is a theoretical study of the problem of formation of noncanonical structures, cruciforms in palindromic regions and the Z form in purine--pyrimidine sequences, in negatively supercoiled DNA. If two such regions, one palindromic and one purine--pyrimidine, are present in the same DNA molecule of a finite length, then transitions between the regular B form and noncanonical structures in these regions will experience a considerable mutual influence. This takes place because both noncanonical structures compete for the same superhelix energy. A special attention is paid to the case when the probability of the Z form formation nonmonotonously depends on the superhelix density. Such a situation is shown to be possible for some specific interrelation between the DNA length, the length of the palindromic region and the length of the purine--pyrimidine region. The calculations show that in this case the Z form is formed first with the increasing superhelix density, that the cruciform structure is formed whereas the purine--pyrimidine region returns into the B form, and finally, the Z form is formed again. The possibility of experimental observation of such unusual behaviour is discussed.     

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.     

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).     

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.     

S-DNA is oversupercoiled DNA with 1.94-2.19 A rise per base pair along duplex axis

Supercoiled pGEMEX DNA with length of 3993 nucleotides was immobilized on four substrates (freshly cleaved mica, standard amino mica, modified amino mica with increased and decreased surface charge density compared with standard amino mica) and it was visualized by atomic force microscopy (AFM) in air. Plectonomically supercoiled DNA molecules as well as single molecules with extremely high level of compaction (i.e. molecules with significantly higher superhelix density values on comparison with previously experimentally measured and theoretically investigated ones) were visualized on modified amino mica which was characterized by increased surface charge density. Distance between base pairs along duplex axis was determined by measurements of contour length of single oversupercoiled DNA molecules. Determined rise per base pair was varied from 1.94 to 2.19 A. These compressed supercoiled DNA molecules like a spring with decreased rise/base pair on comparison with well-known DNA forms were called new DNA form--S-DNA. A model of S-DNA was built. Formation of the S-DNA molecules was suggested to be an intermediate stage on the compaction of the single supercoiled DNA molecules up to the spheroids and minitoroids. Oversupercoiling and further compression of the supercoiled DNA molecules was shown to cause by high surface charge density of amino mica on which DNA molecules were immobilized.     

Post-synthetic acetylation of HMGB1 protein modulates its interactions with supercoiled DNA

High mobility group box (HMGB) proteins 1 and 2 are abundant non-histone nuclear proteins that regulate chromatin structure because of their structure-specific binding to DNA. Here, we have investigated how the post-synthetic acetylation of HMGB1 affects its interaction with negatively supercoiled DNA by employing monoacetylated at Lys2 protein, isolated from butyrate-treated cells. Our data reveal that this modification enhances three reaction parameters: binding affinity, supercoiling activity and capacity to protect the supercoiled DNA from relaxation by topoisomerase I. We show that monoacetylation at Lys2 mimics the effect of acidic tail removal but to a lesser extent thus demonstrating that in vivo acetylated HMGB1 is capable of modulating its interaction with negatively supercoiled DNA.     

DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes

Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.     

The genetic control of DNA supercoiling in Salmonella typhimurium

We have elucidated the genetic control of DNA supercoiling in Salmonella typhimurium. The level of superhelix density is controlled by two classes of genes. The only member of the first class is topA, the structural gene for topoisomerase I. The second class, tos, (topoisomerase one suppressor) consists of at least two genes, one of which is linked to gyrA, the structural gene for the topoisomerase subunit of DNA gyrase. Deletions of topA result in oversupercoiling of plasmid DNA. These mutations do not require the acquisition of second-site compensatory mutations to allow cell growth, in contrast to the situation in Escherichia coli. However, tos mutations, unlinked to topA, have been isolated which reduce plasmid superhelix density. We conclude that the level of DNA supercoiling in S. typhimurium is a dynamic balance between the effects of the gene products of topA (relaxation) and tos (supercoiling) which act independently of each other. Using a variety of combinations of these mutations we have constructed a series of isogenic strains, each of which has a different but precisely defined level of plasmid supercoiling; the series as a whole provides a wide range of supercoiling both above and below the wild-type level.     

Protein motion from non-specific to specific DNA by three-dimensional routes aided by supercoiling

DNA-binding proteins are generally thought to locate their target sites by first associating with the DNA at random and then translocating to the specific site by one-dimensional (1D) diffusion along the DNA. We report here that non-specific DNA conveys proteins to their target sites just as well when held near the target by catenation as when co-linear with the target. Hence, contrary to the prevalent view, proteins move from random to specific sites primarily by three-dimensional (3D) rather than 1D pathways, by multiple dissociation/re-association events within a single DNA molecule. We also uncover a role for DNA supercoiling in target-site location. Proteins find their sites more readily in supercoiled than in relaxed DNA, again indicating 3D rather than 1D routes.