DNA bending,compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea Sulfolobus. Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.     

Probing Rad51-DNA interactions by changing DNA twist

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.     

Thermodynamics of Forming a Parallel DNA Crossover

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX₁ molecule, wherein one crossover is missing between the two helical domains. The JX₁ molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.     

Experimental evaluation of the Liu–Beveridge dinucleotide step model of DNA structure

Methods for predicting DNA curvature have many possible applications. Dinucleotide step models describe DNA shape by characterization of helical twist, deflection angles and the direction of deflection for nearest neighbor base pairs. Liu and Beveridge have extended previous applications of dinucleotide step models with the development and qualitative validation of a predictive method for sequence-dependent DNA curvature (the LB model). We tested whether the LB model accurately predicts experimentally deduced curvature angles and helical repeat parameters for DNA sequences not in its training set, particularly when challenged with quantitative data and subtle sequence phasings. We examined a series of 17 well-characterized DNA sequences to compare electrophoretic and computational results. The LB model is superior to two other models in the prediction of helical repeat parameters. We observed a strong linear correlation between curvature magnitudes predicted using the LB model and those determined by electrophoretic ligation ladder experiments, although the LB model somewhat underestimated apparent curvature. With longer electrophoretic phasing probes the LB model slightly overestimated gel mobility anomalies, with modest deviations in predicted helical repeat parameters. Overall, our analyses suggest that the LB model provides reasonably accurate predictions for the electrophoretic behavior of DNA.     

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.     

The flexibility of DNA double crossover molecules

Double crossover molecules are DNA structures containing two Holliday junctions connected by two double helical arms. There are several types of double crossover molecules, differentiated by the relative orientations of their helix axes, parallel or antiparallel, and by the number of double helical half-turns (even or odd) between the two crossovers. They are found as intermediates in meiosis and they have been used extensively in structural DNA nanotechnology for the construction of one-dimensional and two-dimensional arrays and in a DNA nanomechanical device. Whereas the parallel double helical molecules are usually not well behaved, we have focused on the antiparallel molecules; antiparallel molecules with an even number of half-turns between crossovers (termed DAE molecules) produce a reporter strand when ligated, facilitating their characterization in a ligation cyclization assay. Hence, we have estimated the flexibility of antiparallel DNA double crossover molecules by means of ligation-closure experiments. We are able to show that these molecules are approximately twice as rigid as linear duplex DNA.     

Helical Chirality: a Link between Local Interactions and Global Topology in DNA

DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg²⁺ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology.     

Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure

We describe a model system for study of chromatin structure at levels above that of the nucleosome. A series of fragments with lengths ranging from 172 to 207 bp tandemly repeated three to greater than 50 times was prepared; each repeat contains the region important in forming a positioned core particle on a sea urchin 5S rRNA gene upon in vitro association with histones. The tandemly repeated sequences can be studied as linear DNA fragments or as relaxed or supercoiled circular molecules. A number of criteria indicate that nucleosomes position correctly on all the tandemly repeated elements. Measurement of the change in linking number per core particle led to a value of -1.0. Both length and repeat number dependent changes in conformation of the nucleoproteins are observed. We discuss the possibility that some ordered higher level chromatin structure can form with DNA and core histones alone.     

Oblique section 3-D reconstruction of relaxed insect flight muscle reveals the cross-bridge lattice in helical registration.

In this work we examined the arrangement of cross-bridges on the surface of myosin filaments in the A-band of Lethocerus flight muscle. Muscle fibers were fixed using the tannic-acid-uranyl-acetate, ("TAURAC") procedure. This new procedure provides remarkably good preservation of native features in relaxed insect flight muscle. We computed 3-D reconstructions from single images of oblique transverse sections. The reconstructions reveal a square profile of the averaged myosin filaments in cross section view, resulting from the symmetrical arrangement of four pairs of myosin heads in each 14.5-nm repeat along the filament. The square profiles form a very regular right-handed helical arrangement along the surface of the myosin filament. Furthermore, TAURAC fixation traps a near complete 38.7 nm labeling of the thin filaments in relaxed muscle marking the left-handed helix of actin targets surrounding the thick filaments. These features observed in an averaged reconstruction encompassing nearly an entire myofibril indicate that the myosin heads, even in relaxed muscle, are in excellent helical register in the A-band.     

Colobus type C virus: molecular cloning of unintegrated viral DNA and characterization of the endogenous viral genomes of Colobus.

The unintegrated viral DNA intermediates of colobus type C virus (CPC-1) were isolated from infected human cells that were permissive for viral growth. There were two major species of DNA, linear molecules with two copies of the long terminal repeat and relaxed circles containing only a single long terminal repeat. In addition, there was a minor species (approximately 10%) composed of relaxed circles with two copies of the long terminal repeat. A restriction endonuclease map of the unintegrated DNA was constructed. The three EcoRI fragments of circular CPC-1 DNA were cloned in the EcoRI site of lambda gtWES . lambda B and then subcloned in the EcoRI site of pBR322. Using these subgenomic fragments as probes, we have characterized the endogenous viral sequences found in colobus cellular DNA. They are not organized in tandem arrays, as is the case in some other gene families. The majority of sequences detected in cellular DNA have the same map as the CPC-1 unintegrated DNA at 17 of 18 restriction endonuclease sites. There are, however, other sequences that are present in multiple copies and do not correspond to the CPC-1 map. They do not contain CPC-1 sequences either in an altered form or fused to common nonviral sequences. Instead, they appear to be derived from a distinct family of sequences that is substantially diverged from the CPC-1 family. This second family of sequences, CPC-2, is also different from the sequences related to baboon endogenous type C virus that forms a third family of virus-related sequences in the colobus genome.     

Organization of internucleosomal DNA in rat liver chromatin

A detailed analysis of the length distribution of DNA in nucleosome dimers trimmed with exonuclease III and S1 nuclease suggests that the previously described variation of internucleosomal distance in rat liver occurs, at least for a subset of the nucleosomes, by integral multiples of the helical repeat of the DNA. Results obtained upon digestion of chromatin with DNase II further suggest that lengths of internucleosomal DNA are integral multiples of the helical repeat of the DNA plus approximately 5 bp. Restraints imposed by these features on the arrangement of nucleosomes along the fiber are discussed.     

Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA

Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.     

No braiding of Holliday junctions in positively supercoiled DNA molecules

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.     

Patterson methods in fibre diffraction analysis.

The potential of the Patterson methods for X-ray diffraction studies of textures is examined in DNA fibres. Patterson analysis, which is rarely used in these situations, is shown to yield important information on the preliminary interpretation of diffraction patterns and to increase the reliability of the three-dimensional structural pattern obtained for polymeric molecules. We also show how the screw symmetry of helical molecules can be used to calculate their electron density by means of the three-dimensional Patterson function.     

The effect of ionic conditions on DNA helical repeat, effective diameter and free energy of supercoiling.

We determined the free energy of DNA supercoiling as a function of the concentration of magnesium and sodium chloride in solution by measuring the variance of the equilibrium distribution of DNA linking number,<(DeltaLk)2>. We found that the free energy of supercoiling changed >1.5-fold over the range of ionic conditions studied. Comparison of the experimental results with those of computer simulations showed that the ionic condition dependence of<(DeltaLk)2>is due mostly to the change in DNA effective diameter, d, a parameter characterizing the electrostatic interaction of DNA segments. To make this comparison we determined values of d under all ionic conditions studied by measuring the probability of knot formation during random cyclization of linear DNA molecules. From the topoisomer distributions we could also determine the changes in DNA helical repeat, gamma, in mixed NaCl/MgCl2 solutions. Both gamma and d exhibited a complex pattern of changes with changing ionic conditions, which can be described in terms of competition between magnesium and sodium ions for binding to DNA.     

Structural changes that occur in scallop myosin filaments upon activation

Myosin filaments isolated from scallop striated muscle have been activated by calcium-containing solutions, and their structure has been examined by electron microscopy after negative staining. The orderly helical arrangement of myosin projections characteristic of the relaxed state is largely lost upon activation. The oblique striping that arises from alignment of elongated projections along the long-pitched helical tracks is greatly weakened, although a 145 A axial periodicity is sometimes partially retained. The edges of the filaments become rough, and the myosin heads move outwards as their helical arrangement becomes disordered. Crossbridges at various angles appear to link thick and thin filaments after activation. The transition from order to disorder is reversible and occurs over a narrow range of free calcium concentration near pCa 5.7. Removal of nucleotide, as well as dissociation of regulatory light chains, also disrupts the ordered helical arrangement of projections. We suggest that the relaxed arrangement of the projections is probably maintained by intermolecular interactions between myosin molecules, which depend on the regulatory light chains. Calcium binding changes the interactions between light chains and the rest of the head, activating the myosin molecule. Intermolecular contacts between molecules may thus be altered and may propagate activation cooperatively throughout the thick filament.     

Characterization of inherent curvature in DNA lacking polyadenine runs.

Sequence-directed DNA curvature is most commonly associated with AA dinucleotides in the form of polyadenine runs. We demonstrate inherent curvature in DNA which lacks AA/TT dinucleotides using the criteria of polyacrylamide gel mobility and efficiency of DNA cyclization. These studies are based upon two 21-base pair synthetic DNA fragments designed to exhibit fixed curvature according to deflections made to the helical axis by non-AA dinucleotide stacks. Repeats of these sequences display anomalously slow migration in polyacrylamide gels. Moreover, both sequences describe helical conformations that are closed into circles by DNA ligase at much smaller sizes than is typical of nondeformed DNA. Chemical cleavage of these DNA molecules with hydroxyl radical is also consistent with local variation in helical conformation at specific dinucleotide steps.     

The supercoil-stabilised cruciform of ColE1 is hyper-reactive to osmium tetroxide

Supercoiled pColIR215 contains a site of pronounced hyper-reactivity towards modification by osmium tetroxide, a reagent known to be single-strand-selective. The site of hypersensitivity has been mapped to the ColE1 inverted repeat, believed to extrude a cruciform in supercoiled DNA. Linear or relaxed plasmids are not modified by the reagent. We conclude that cruciform formation is responsible for the site-selective modification. Fine mapping of the modification site as a function of time has revealed that the initial reaction occurs at the centre of the inverted repeat, i.e., the unpaired loop of the cruciform, but that the modification region rapidly expands outwards from this point.