Supramolecular polymorphism of DNA in non-cationic L(alpha) lipid phases

The structure of a complex between hydrated DNA and a non-cationic lipid is studied, including its phase diagram. The complex is spontaneously formed by adding DNA fragments (ca. 150 base pairs in length) to non-cationic lipids and water. The self-assembly process often leads to highly ordered structures. The structures were studied by combining X-ray scattering, fluorescence and polarized microscopy, as well as freeze-fracture experiments with transmission electron microscopy. We observe a significant increase of the smectic order as DNA is incorporated into the water layers of the lamellar host phase, and stabilization of single phase domains for large amounts of DNA. The effect of confinement on DNA ordering is investigated by varying the water content, following three dilution lines. A rich polymorphism is found, ranging from weakly correlated DNA-DNA in-plane organizations to highly ordered structures, where transmembrane correlations lead to the formation of columnar rectangular and columnar hexagonal superlattices of nucleotides embedded between lipid lamellae. From these observations, we suggest that addition of DNA to the lamellar phase significantly restricts membrane fluctuations above a certain concentration and helps the formation of the lipoplex. The alteration of membrane steric interactions, together with the appearance of interfacial interactions between membranes and DNA molecules may be a relevant mechanism for the emergence of highly ordered structures in the concentrated regime.

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases.

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.

The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix. Possible implications for DNA structure in vivo.

A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules. It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe. When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation. In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other. This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA. In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions. Possible implications of this observed structural transition in DNA are discussed.

DNA supercoiling and its role in DNA decatenation and unknotting.

Chromosomal and plasmid DNA molecules in bacterial cells are maintained under torsional tension and are therefore supercoiled. With the exception of extreme thermophiles, supercoiling has a negative sign, which means that the torsional tension diminishes the DNA helicity and facilitates strand separation. In consequence, negative supercoiling aids such processes as DNA replication or transcription that require global- or local-strand separation. In extreme thermophiles, DNA is positively supercoiled which protects it from thermal denaturation. While the role of DNA supercoiling connected to the control of DNA stability, is thoroughly researched and subject of many reviews, a less known role of DNA supercoiling emerges and consists of aiding DNA topoisomerases in DNA decatenation and unknotting. Although DNA catenanes are natural intermediates in the process of DNA replication of circular DNA molecules, it is necessary that they become very efficiently decatenated, as otherwise the segregation of freshly replicated DNA molecules would be blocked. DNA knots arise as by-products of topoisomerase-mediated intramolecular passages that are needed to facilitate general DNA metabolism, including DNA replication, transcription or recombination. The formed knots are, however, very harmful for cells if not removed efficiently. Here, we overview the role of DNA supercoiling in DNA unknotting and decatenation.

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination.

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

Opposite effect of counterions on the persistence length of nicked and non-nicked DNA.

Using cryo-electron microscopy we reconstructed the three-dimensional trajectories adopted in cryovitrified solutions by double-stranded DNA molecules in which the backbone of one strand lacked a phosphate at regular intervals of 20 nucleotides. The shape of such nicked DNA molecules was compared with that of DNA molecules with exactly the same sequence but without any single-stranded scissions. Upon changing the salt concentration we observed opposite effects of charge neutralization on nicked and non-nicked DNA. In low salt solutions (10 mM Tris-HCl, 10 mM NaCl) the applied dense nicking caused ca 3.5-fold reduction of the DNA persistence length as compared with non-nicked DNA. Upon increasing the salt concentration (to 150 mM NaCl and 10 mM MgCl2) the persistence length of non-nicked DNA appreciably decreased while that of nicked DNA molecules increased by a factor of 2.

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.

Geometry and physics of catenanes applied to the study of DNA replication.

The concept of ideal geometric configurations was recently applied to the classification and characterization of various knots. Different knots in their ideal form (i.e., the one requiring the shortest length of a constant-diameter tube to form a given knot) were shown to have an overall compactness proportional to the time-averaged compactness of thermally agitated knotted polymers forming corresponding knots. This was useful for predicting the relative speed of electrophoretic migration of different DNA knots. Here we characterize the ideal geometric configurations of catenanes (called links by mathematicians), i.e., closed curves in space that are topologically linked to each other. We demonstrate that the ideal configurations of different catenanes show interrelations very similar to those observed in the ideal configurations of knots. By analyzing literature data on electrophoretic separations of the torus-type of DNA catenanes with increasing complexity, we observed that their electrophoretic migration is roughly proportional to the overall compactness of ideal representations of the corresponding catenanes. This correlation does not apply, however, to electrophoretic migration of certain replication intermediates, believed up to now to represent the simplest torus-type catenanes. We propose, therefore, that freshly replicated circular DNA molecules, in addition to forming regular catenanes, may also form hemicatenanes.

Determination of DNA persistence length by cryo-electron microscopy. Separation of the static and dynamic contributions to the apparent persistence length of DNA.

Axial deflection of DNA molecules in solution results from thermal motion and intrinsic curvature related to the DNA sequence. In order to measure directly the contribution of thermal motion we constructed intrinsically straight DNA molecules and measured their persistence length by cryo-electron microscopy. The persistence length of such intrinsically straight DNA molecules suspended in thin layers of cryo-vitrified solutions is about 80 nm. In order to test our experimental approach, we measured the apparent persistence length of DNA molecules with natural "random" sequences. The result of about 45 nm is consistent with the generally accepted value of the apparent persistence length of natural DNA sequences. By comparing the apparent persistence length to intrinsically straight DNA with that of natural DNA, it is possible to determine both the dynamic and the static contributions to the apparent persistence length.

The ATPase activity of RecA is needed to push the DNA strand exchange through heterologous regions.

The role of ATP hydrolysis during the RecA-mediated recombination reaction is addressed in this paper. Recent studies indicated that the RecA-promoted DNA strand exchange between completely homologous double- and single-stranded DNA can be very efficient in the absence of ATP hydrolysis. In this work we demonstrate that the energy derived from the ATP hydrolysis is strictly needed to drive the DNA strand exchange through the regions where the interacting DNA molecules are not in a homologous register. Therefore, in addition to the role of the ATP hydrolysis in promoting the dissociation of RecA from the products of the recombination reaction, as described earlier, ATP hydrolysis also plays a crucial role in the actual process of strand exchange, provided that the lack of homologous register obstructs the process of branch migration.

RecA-DNA helical filaments in genetic recombination.

Paul Howard-Flanders et al proposed a molecular model of RecA-mediated recombination reaction six years ago. How does this model stand at present? In answering this question, we focus on two leading ideas of the original model, namely the proposal of the coaxial arrangement of the aligned DNA molecules within helical RecA filaments and the proposal of the ATP independence of the pairing stage of the recombination reaction. Results obtained after the model was proposed are reviewed and compared with these original assumptions and postulates of the model. EM visualization of recombining DNA molecules, studies of the energetics of the RecA-mediated recombination reaction and biochemical analysis of deproteinized joint molecules are fully consistent with a triple-stranded DNA arrangement during the RecA-mediated recombination reaction and demonstrate the ATP independence of the pairing stage of the reaction.

DNA supercoiling inhibits DNA knotting.

Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecular passages between segments of DNA molecules use the energy of ATP hydrolysis to select passages that lead to unknotting rather than to the formation of knots. Using numerical simulations, we identify here another mechanism by which topoisomerases can keep the knotting level low. We observe that DNA supercoiling, such as found in bacterial cells, creates a situation where intramolecular passages leading to knotting are opposed by the free-energy change connected to transitions from unknotted to knotted circular DNA molecules.

Time-Lapse AFM Imaging of DNA Conformational Changes Induced by Daunorubicin.

Cancer is a major health issue that absorbs the attention of a large part of the biomedical research. Intercalating agents bind to DNA molecules and can inhibit their synthesis and transcription; thus, they are increasingly used as drugs to fight cancer. In this work, we show how atomic force microscopy in liquid can characterize, through time-lapse imaging, the dynamical influence of intercalating agents on the supercoiling of DNA, improving our understanding of the drug's effect.

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases.

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.

Simulations of electrophoretic collisions of DNA knots with gel obstacles

Gel electrophoresis can be used to separate nicked circular DNA molecules of equal length but forming different knot types. At low electric fields, complex knots drift faster than simpler knots. However, at high electric field the opposite is the case and simpler knots migrate faster than more complex knots. Using Monte Carlo simulations we investigate the reasons of this reversal of relative order of electrophoretic mobility of DNA molecules forming different knot types. We observe that at high electric fields the simulated knotted molecules tend to hang over the gel fibres and require passing over a substantial energy barrier to slip over the impeding gel fibre. At low electric field the interactions of drifting molecules with the gel fibres are weak and there are no significant energy barriers that oppose the detachment of knotted molecules from transverse gel fibres.