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Pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. While in some organisms meiotic pairing requires programmed DNA breakage and recombination, in many others it engages homologous chromosomes that remain apparently intact. The mechanistic nature of such recombination-independent pairing represents a fundamental question in molecular genetics. Using ‘meiotic silencing by unpaired DNA’ (MSUD) in Neurospora crassa as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesin complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be co-linear and must contain short tracts of sequence identity spaced apart with a periodicity of 21 or 22 base-pairs. By using these values as a guiding parameter in all-atom molecular modeling, we discover that homologous double-stranded DNA molecules can associate by forming quadruplex-based contacts with an interval of 2.5 helical turns, which requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. These results (i) reconcile genetic and biophysical lines of evidence for the existence of direct homologous dsDNA-dsDNA pairing, (ii) identify a role for this process in initiating post-transcriptional silencing, and (iii) suggest that chromosomes are cross-matched in meiosis by a precise mechanism that operates on intact double-stranded DNA molecules.

The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using "meiotic silencing by unpaired DNA" (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)-dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.

In cells, DNA is routinely subjected to significant levels of bending and twisting. In some cases, such as under physiological levels of supercoiling, DNA can be so highly strained, that it transitions into non-canonical structural conformations that are capable of relieving mechanical stress within the template. DNA minicircles offer a robust model system to study stress-induced DNA structures. Using DNA minicircles on the order of 100 bp in size, we have been able to control the bending and torsional stresses within a looped DNA construct. Through a combination of cryo-EM image reconstructions, Bal31 sensitivity assays and Brownian dynamics simulations, we have been able to analyze the effects of biologically relevant underwinding-induced kinks in DNA on the overall shape of DNA minicircles. Our results indicate that strongly underwound DNA minicircles, which mimic the physical behavior of small regulatory DNA loops, minimize their free energy by undergoing sequential, cooperative kinking at two sites that are located about 180 degrees apart along the periphery of the minicircle. This novel form of structural cooperativity in DNA demonstrates that bending strain can localize hyperflexible kinks within the DNA template, which in turn reduces the energetic cost to tightly loop DNA.

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.

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.

Circular DNA molecules in vivo form catenanes and knots during such processes as replication or recombination. In addition, DNA molecules are often subjected to a torsional tension, which results in their supercoiling. The interplay between catenation, knotting and supercoiling leads to unexpected conformational changes of entire DNA molecules, and this has interesting physical and biological consequences. We show in this chapter how modelling DNA as a semi-flexible ribbon can be applied to get information about the molecular conformation of DNA molecules with complex topologies. In particular, our results highlight the importance of the chirality of knots and catenanes in the structural changes induced by DNA supercoiling. For example, strongly linked right-handed toroidal DNA catenanes undergo a specific folding that can be reversed by the introduction of negative supercoiling in each chain, or the shape of negatively supercoiled DNA trefoil knots depends on their chirality. In each case, we perform dynamical simulations including hydrodynamics, to investigate the consequences of these structural changes on the sedimentation and gel electrophoretic behaviour of the modelled knotted and catenated DNA molecules.

We have characterized the polymer physics of single-stranded DNA (ssDNA) using atomic force microscopy. The persistence length l(p) of circular ssDNA adsorbed on a modified graphite surface was determined independently of secondary structure. At a very low ionic strength we obtained l(p)=9.1 nm from the bond correlation function. Increasing the salt concentration lead to a decrease in l(p); at 1 mM NaCl we found l(p)=6.7 nm, while at 10 mM NaCl a value l(p)=4.6 nm was obtained. The persistence length was also extracted from the root-mean-square end-to-end distance and the end-to-end distance distribution function. Finally, we have investigated the scaling behavior using the two latter quantities, and found that on long length scales ssDNA behaves as a two-dimensional self-avoiding walk.

12 páginas, 8 figuras -- PAGS nros. 3845-3853, The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo., Spanish Ministerio de Ciencia e Innovacion[BIO2005-02224, BFU2008-00408/BMC, BFU2007-62670]; Swiss National Science Foundation[3100A0-116275]

The conformation of circular DNA molecules of various lengths adsorbed in a 2D conformation on a mica surface is studied. The results confirm the conjecture that the critical exponent nu is topologically invariant and equal to the self-avoiding walk value (in the present case nu=3/4), and that the topology and dimensionality of the system strongly influence the crossover between the rigid regime and the self-avoiding regime at a scale L approximately 7l{p}. Additionally, the bond correlation function scales with the molecular length L as predicted. For molecular lengths Lor=5l{p}, circular DNA behaves like a stiff molecule with an approximately elliptic shape.

We study the behavior of single-stranded DNA (ssDNA) in the presence of well-known drugs with either an intercalating binding mode, such as daunorubicin, actinomycin D, and chloroquine, or a minor groove binding mode, such as netropsin and berenil, by atomic force microscopy (AFM). At very low salt conditions, ssDNA molecules adopt an unstructured conformation without secondary structures. We observe that under these conditions additions of drugs that bind to double-stranded DNA ( dsDNA) promote the formation of secondary structures in ssDNA. Furthermore, with an increase of concentration of the drugs, the extension as well as the thermal stabilization of these hairpins was observed.

Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg2+, by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.

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 bond orientational correlation function (BCF) of a semiflexible ring polymer on a flat surface is studied theoretically. For a stiff chain, we give an exact analytic form of BCF with perturbation calculations. For a chain sufficiently longer than its persistence length, the conventional exponential decay vanishes and a long-range order along the chain contour appears. We demonstrate that the bond orientational correlation satisfies the scaling properties, and construct an interpolating formula for its universal curve that encompasses the short- and large-distance behaviors. Our analytical findings are confirmed by extensive Langevin dynamics simulations, and are in excellent agreement with recent experimental data obtained from DNA molecules imaged by atomic force microscopy without any fitting parameters.

Two-dimensional semiflexible polymer rings are studied both by imaging circular DNA adsorbed on a mica surface and by Monte Carlo simulations of phantom polymers as well as of polymers with finite thickness. Comparison of size and shape of the different models over the full range of flexibilities shows that excluded volume caused by finite thickness induces an anisotropic increase of the main axes of the conformations, a change of shape, accomplished by an enhanced correlation along the contour., 5 pages, 4 figures, version as published in Nano Letters

Replication of circular DNA proceeds through a stage of multiply interlinked catenanes that have to be rapidly spatially separated. In addition, knotting of circular DNA has also to be avoided. In bacteria, topology simplification requires participation of two type II topoisomerases: gyrase and topo IV. Several simulation approaches were applied to explain the very efficient topology simplification in that system. Mainly two strategies were explored: in the first one, the system follows its free energy gradient influenced by supercoiling, and in the second one, specific geometrical rules are defined for the selection of strand passages (hooking, chirality). The Monte-Carlo methods usually used to estimate the efficiency of these strategies do not allow to follow DNA topology simplification dynamically, to evaluate its speed, for example. To overcome this limitation, we simulated DNA unknotting and decatenation by Brownian dynamics, which allows for a natural integration of the strategies mentioned above. By following the topological state of the simulated DNA chains (see figure), we show that the combination of supercoiling and local geometrical selection rules provides an important drive for unknotting and decatenation, especially at low topological complexity.View Large Image | View Hi-Res Image | Download PowerPoint Slide