Strand Crossovers Research Articles

Triple-Stranded DNA As a Structural Element in DNA Origami.

Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex-triplex DNA motifs as pH-dependent building blocks in DNA origami. We investigate design rules for incorporating triplex forming oligonucleotides and noncanonical duplex-triplex crossovers in multilayer DNA origami objects. We use single-particle cryoelectron microscopy to elucidate the structural basis of triplex domains and of duplex-triplex crossovers. We find that duplex-triplex crossovers can complement and fully replace the canonical duplex-duplex crossovers within DNA origami objects, for example, to increase the crossover density for potentially greater rigidity and reduced interhelical spacing, and to create connections at sites where conventional crossovers may be undesirable. We also show the pH-induced formation of a DNA origami object stabilized entirely by triplex-mediated strand crossovers.

Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double-strand breaks but form by different mechanisms: noncrossovers by synthesis-dependent strand annealing and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes involving branch migration as an integral feature, one that can be separated from repair of the break itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

Lattice models and Monte Carlo methods for simulating DNA origami self-assembly.

The optimal design of DNA origami systems that assemble rapidly and robustly is hampered by the lack of a model for self-assembly that is sufficiently detailed yet computationally tractable. Here, we propose a model for DNA origami that strikes a balance between these two criteria by representing these systems on a lattice at the level of binding domains. The free energy of hybridization between individual binding domains is estimated with a nearest-neighbour model. Double helical segments are treated as being rigid, but we allow flexibility at points where the backbone of one of the strands is interrupted, which provides a reasonably realistic representation of partially and fully assembled states. Particular attention is paid to the constraints imposed by the double helical twist, as they determine where strand crossovers between adjacent helices can occur. To improve the efficiency of sampling configuration space, we develop Monte Carlo methods for sampling scaffold conformations in near-assembled states, and we carry out simulations in the grand canonical ensemble, enabling us to avoid considering states with unbound staples. We demonstrate that our model can quickly sample assembled configurations of a small origami design previously studied with the oxDNA model, as well as a design with staples that span longer segments of the scaffold. The sampling ability of our method should allow for good statistics to be obtained when studying the assembly pathways and is suited to investigating, in particular, the effects of design and assembly conditions on these pathways and their resulting final assembled structures.

Dimensions and Global Twist of Single-Layer DNA Origami Measured by Small-Angle X-ray Scattering.

The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.

Electrotransfection of Polyamine Folded DNA Origami Structures.

DNA origami structures are artificial molecular nanostructures in which DNA double helices are forced into a closely packed configuration by a multitude of DNA strand crossovers. We show that three different types of origami structures (a flat sheet, a hollow tube, and a compact origami block) can be formed in magnesium-free buffer solutions containing low (<1 mM) concentrations of the condensing agent spermidine. Much like in DNA condensation, the amount of spermidine required for origami folding is proportional to the DNA concentration. At excessive amounts, the structures aggregate and precipitate. In contrast to origami structures formed in conventional buffers, the resulting structures are stable in the presence of high electric field pulses, such as those commonly used for electrotransfection experiments. We demonstrate that spermidine-stabilized structures are stable in cell lysate and can be delivered into mammalian cells via electroporation.

Base-Pair Level Analysis of DNA Four-Way Junction Structure and Dynamics

Immobile four-way junctions (4WJs) form a core structural motif of structured DNA assemblies. Understanding the sequence-level impact of four-way junctions on their structure, stability, and flexibility plays an important role in achieving Angstrom-level spatial control over nucleic acid nanodevices. 4WJs may exist in one of two stacked conformational isomers that are preferentially adopted when free in solution but forced into one or the other configuration in programmed multi-junction assemblies. It has been shown experimentally that the base sequences around the sites of strand crossover in these junctions have a significant impact on the conformational preferences of the two isomers.1,2 In particular, some sequences exhibit significant bias for one isomeric state, whereas others have equal preference between the two isomers. Here, we use explicit solvent and counterion molecular dynamics simulations to explore the base-pair level interactions that give rise to 4WJ conformational states using the classic Seeman J1 junction3 system as a point of reference. A pseudo-alchemical path approach is used to investigate how nicks placed at the site of the strand crossovers impact B-form DNA and crossover geometry and stability, and their dependence on core 4WJ sequence. Free energy calculations and analysis of the base-pair level degrees of freedom4 for each step along this path reveal the structural origin of the significant impact of base sequence on local structure, and suggest how these in turn impact global junction behaviour.

Dependence of the contact resistance on the design of stranded conductors.

During the manufacturing process multi-strand conductors are subject to compressive force and rotation moments. The current distribution in the multi-strand conductors is not uniform and is controlled by the transverse resistivity. This is mainly determined by the contact resistance at the strand crossovers and inter-strand contact resistance. The surface layer properties, and in particular the crystalline structure and degree of oxidation, are key parameters in determining the transverse resistivity. The experimental set-ups made it possible to find the dependence of contact resistivity as a function of continuous working stresses and cable design. A study based on measurements and numerical simulation is made to identify the contact resistivity functions.

Backbone-Branched DNA Building Blocks for Facile Angular Control in Nanostructures

Nanotechnology based on the highly specific pairing of nucleobases in DNA has been used to generate a wide variety of well-defined two- and three-dimensional assemblies, both static and dynamic. However, control over the junction angles to achieve them has been limited. To achieve higher order assemblies, the strands of the DNA duplex are typically made to deviate at junctions with configurations based on crossovers or non-DNA moieties. Such strand crossovers tend to be intrinsically unstructured with the overall structural rigidity determined by the architecture of the nanoassembly, rather than the junction itself. Specific approaches to define nanoassembly junction angles are based either on the cooperative twist- and strain-promoted tuning of DNA persistence length leading to bent DNA rods for fairly large nano-objects, or de novo synthesis of individual junction inserts that are typically non-DNA and based on small organic molecules or metal-coordinating ligand moieties. Here, we describe a general strategy for direct control of junction angles in DNA nanostructures that are completely tunable about the DNA helix. This approach is used to define angular vertices through readily accessible backbone-branched DNAs (bbDNAs). We demonstrate how such bbDNAs can be used as a new building block in DNA nanoconstruction to obtain well-defined nanostructures. Angular control through readily accessible bbDNA building block provides a general and versatile approach for incorporating well-defined junctions in nanoconstructs and expands the toolkit toward achieving strain free, highly size- and shape-tunable DNA based architectures.

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg2+ cations with Ca2+ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.

Progress in the Manufacture of Long Length YBCO Roebel Cables

We have manufactured lengths of up to 10 m of Roebel cable from wide strips of second generation YBCO wire. The wide strips are first screened using magnetic scanning to check for superconductor defects to avoid incorporating poor quality wire into the cable. Manufacturing issues such as the long length registration of the strand crossovers and the maximum yield from wide strip is discussed. Differences in cutting and winding due to different tape substrate materials are shown to be minimal. We report electrical measurements on samples of HTS Roebel cable. Insulation which protects the assembled conductors from environmental degradation is reported.

Structural effects of nogalamycin, an antibiotic antitumour agent, on DNA

The structural changes of DNA, induced by the antitumour antibiotic nogalamycin, have been studied by atomic force microscopy (AFM). The transformation in the tertiary structure of 4361 bp long plasmid pBR322 DNA, after incubation with nogalamycin at 37 °C, has been monitored at the single molecule level. The AFM topographs of free DNA and the DNA–nogalamycin complex, incubated for 6, 12, 24, 36 and 48 h, reveal a gradual change from the circular supercoiled form having strand crossovers to the more compact plectonemic superhelix. With increasing incubation time, the extent of plectonemic coiling increases, indicating increasing level of drug binding via intercalative mode. Supportive evidences are obtained from the CD and UV–vis spectroscopic studies. To our knowledge, this is the first report on an AFM imaging study of the effects of nogalamycin, an anthracyclin intercalator, on DNA.

A solution for transverse load degradation in ITERNb3Sn CICCs: verification of cabling effect on Lorentz force response

We present the latest results of the novel model for transverse electromagnetic load optimization(TEMLOP) especially developed for the ITER type of cable-in-conduit conductors (CICCs). TheNb3Sn CICCs for the International Thermonuclear Experimental Reactor (ITER) showed a substantialdegradation in their performance correlated with increasing electromagnetic load. Not only dothe differences in the thermal contraction of the composite materials affect the critical current(Ic) and temperature margin, but electromagnetic forces cause asignificant transverse strand contact and bending strain in theNb3Sn layers, resulting in localized filament cracking and permanent degradation.The most essential feature of the a priori TEMLOP predictions presented in May 2006 isthat the severe degradation in CICCs can be improved greatly and straightforwardly byincreasing the pitch length in subsequent cabling stages and by reducing the void fraction.These corrective measures give more support to the strands, sufficiently reduce the strain,and therefore avoid filament damage at the strand crossover points in the cables. It was thefirst time that an increase of the cable twist pitches has been proposed and noexperimental evidence was available at that time. A full-size European prototypeTF conductor sample (TFPRO-2), manufactured in autumn 2006, was adaptedaccording to this new insight and tested in April 2007 in SULTAN for experimentalvalidation of the predictions. The results were outstanding: for the first time anNb3Sn CICCconductor achieved the performance that can be expected based on the single-strand properties, withhigh n value and no sign of degradation.As input, besides the cable properties, the model directly uses the measured data fromsingle strands under uni-axial stress and strain, periodic bending and contact loads. Therecent test results of the ITER OST strands used for the manufacture of the TFPRO-2obtained with the TARSIS set-up are presented. With these most recent strand results, themodel substantiates that not only strand bending is causing degradation but,depending on the strand and cable layout, the strand contact stress can also play acritical role. TEMLOP demonstrates that the twist pitch scheme and void fraction,of the proposed ITER reference TF conductor layout with a first-stage triplettwist pitch of 45 mm, turns out to be practically a worst-case scenario. It is alsoshown that shorter pitches can lead to an improvement but this requires moreNb3Sn material per metre composite conductor. However, it has been experimentally proven nowthat the proposed changes recover the ITER TF conductor operational margin up to theexpected strand performance. The ITER TF conductor specification is being adapted nowand it becomes possible to gain significant savings on the strand design, as degradation nolonger needs to be compensated for.

Spatial Periodic Bending and Critical Current of Bronze and PIT ${\rm Nb}_{3}{\rm Sn}$ Strands in a Steel Tube

We measured the influence of spatial periodic bending on the critical current (I <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">c</sub> ) of Nb <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> Sn strands that were tightly swaged in a steel tube, imposing a compressive strain, in attempt to simulate the conditions representative for an ITER (international thermonuclear experimental reactor) cable in conduit conductor (CICC). The huge electromagnetic transverse force causes both transverse compressive strain in the strand crossover contacts and bending strain on top of the strain induced by the differential thermal contraction of the various conductor components. For a CICC with stainless steel or Incoloy conduit, the effect of the thermal cool-down strain on the reduction in performance due to periodic bending strain is unknown. We performed periodic bending tests in the TARSIS facility (test arrangement for strain influence on strands) on three samples. The degradation of the I <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">c</sub> due to bending appears to be much more severe for a strand swaged in a stainless steel tube than straightforward simulated by available models assuming simply axial strain dependency. It is confirmed by AC loss measurements that the extra degradation can not be attributed to a change in matrix transverse resistivity, possibly affecting the current transfer length, so we believe this is related to the three-dimensional strain state of the Nb <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> Sn layer.

The voltage/current characteristic (n index) of the cable-in-conduit conductors for fusion

The index n of the voltage-current characteristic of large superconductors is a crucial parameter to assess the allowed range for coil operation. In the tests of the Insert coils at the CSMC as well as in the short sample test in the SULTAN facility, it is observed that the index n dramatically decreases from the Nb/sub 3/Sn strands extracted from cable-in-conduit provide the evidence of a permanent performance drop in the strands, likely due to the transverse load accumulation at the strand crossovers in the cable. This effect is not observed in NbTi conductors. The immediate implications for the Nb/sub 3/Sn conductor design are discussed and layout modifications to mitigate the effect are proposed.

Interaction in vitro of type III intermediate filament proteins with supercoiled plasmid DNA and modulation of eukaryotic DNA topoisomerase I and II activities.

To further characterize the interaction of cytoplasmic intermediate filament (cIF) proteins with supercoiled (sc)DNA, and to support their potential function as complementary nuclear matrix proteins, the type III IF proteins vimentin, glial fibrillary acidic protein, and desmin were analyzed for their capacities to interact with supercoiled plasmids containing a bent mouse gamma-satellite insert or inserts capable of non-B-DNA transitions into triplex, Z, and cruciform DNA, that is, DNA conformations typically bound by nuclear matrices. While agarose gel electrophoresis revealed a rough correlation between the superhelical density of the plasmids and their affinity for cIF proteins as well as cIF protein-mediated protection of the plasmid inserts from S1 nucleolytic cleavage, electron microscopy disclosed binding of the cIF proteins to DNA strand crossovers in the plasmids, in accordance with their potential to interact with both negatively and positively supercoiled DNA. In addition, the three cIF proteins were analyzed for their effects on eukaryotic DNA topoisomerases I and II. Possibly because cIF proteins interact with the same plectonemic and paranemic scDNA conformations also recognized by topoisomerases, but select the major groove of DNA for binding in contrast to topoisomerases that insert into the minor groove, the cIF proteins were able to stimulate the enzymes in their supercoil-relaxing activity on both negatively and positively supercoiled plasmids. The stimulatory effect was considerably stronger on topoisomerase I than on topoisomerase II. Moreover, cIF proteins assisted topoisomerases I and II in overwinding plasmid DNA with the formation of positive supercoils. Results obtained with the N-terminal head domain of vimentin harboring the DNA binding region and terminally truncated vimentin proteins indicated the involvement of both protein-DNA and protein-protein interactions in these activities. Based on these observations, it seems conceivable that cIF proteins participate in the control of the steady-state level of DNA superhelicity in the interphase nucleus in conjunction with such topoisomerase-controlled processes as DNA replication, transcription, recombination, maintenance of genome stability, and chromosome condensation and segregation.

The Inherent Properties of DNA Four-way Junctions: Comparing the Crystal Structures of Holliday Junctions

Holliday junctions are four-stranded DNA complexes that are formed during recombination and related DNA repair events. Much work has focused on the overall structure and properties of four-way junctions in solution, but we are just now beginning to understand these complexes at the atomic level. The crystal structures of two all-DNA Holliday junctions have been determined recently from the sequences d(CCGGGACCGG) and d(CCGGTACCGG). A detailed comparison of the two structures helps to distinguish distortions of the DNA conformation that are inherent to the cross-overs of the junctions in this crystal system from those that are consequences of the mismatched dG·dA base-pair in the d(CCGGGACCGG) structure. This analysis shows that the junction itself perturbs the sequence-dependent conformational features of the B-DNA duplexes and the associated patterns of hydration in the major and minor grooves only minimally. This supports the idea that a DNA four-way junction can be assembled at relatively low energetic cost. Both structures show a concerted rotation of the adjacent duplex arms relative to B-DNA, and this is discussed in terms of the conserved interactions between the duplexes at the junctions and further down the helical arms. The interactions distant from the strand cross-overs of the junction appear to be significant in defining its macroscopic properties, including the angle relating the stacked duplexes across the junction.

Electromagnetic and mechanical AC loss of an ITER TF model coil conductor (DP4) under transverse cyclic loading

Energising a coil results in a transverse force on the strands pushing the cable towards one side of the jacket. This load causes a transverse compressive strain in strands and in particular in strand crossover points. Besides this, contact surfaces interfere by micro-sliding resulting in friction and anomalous contact resistance behaviour versus force. Two Central Solenoid Model Coil conductors have been tested previously in a cryogenic press and now the experimental results are presented for the Toroidal Field Model Coil (TFMC) conductor (DP4). The press can transmit a variable (cyclic) force of at least 650 kN/m directly to a cable section of 400 mm at 4.2 K. The magnetisation of the conductor and the interstrand resistance (R/sub c/) between various strands inside the cable can be measured by varying pressure. The force on the cable and the displacement are monitored simultaneously in order to determine the effective cable Young's modulus and the mechanical heat generation due to friction and deformation. The mechanical heat generation, the coupling loss time constant n/spl tau/ and the R/sub c/ of the full-size ITER TFMC conductor have been studied under load up to 40 full loading cycles. The evolution of R/sub c/ is comparable to the behaviour found for the CS Model Coil type of conductors. A significant decrease of the cable coupling current time constant, n/spl tau/ and mechanical heat generation after cyclic loading is found.

A new Holliday junction resolving enzyme from Schizosaccharomyces pombe that is homologous to CCE1 from Saccharomyces cerevisiae

The resolution of Holliday junctions is a critical stage in recombination. We describe the identification and initial biochemical characterisation of a new Holliday junction resolvase from Schizosaccharomyces pombe. Resolvase activity was initially detected in partially purified cell-free extracts of S. pombe. Resolution of X-junction DNA occurred by the introduction of symmetrical cuts in strands of the same polarity. All cuts occurred 3′ of thymine nucleotides with a possible preference for cleavage one nucleotide 3′ from the point of strand crossover. During the course of these studies, a potential S. pombe homologue of the Saccharomyces cerevisiae Cruciform Cutting Endonuclease I was identified in the database ( SpCCE1). The gene was cloned by PCR, overexpressed in Escherichia coli and its product purified as a His-tagged fusion protein. Purified SpCCE1 binds to X-junctions in a structure-specific manner and resolves them to nicked linear duplex products that are repairable by DNA ligase. SpCCE1 cuts X-junctions in precisely the same way as the resolvase activity from partially purified extracts of S. pombe, indicating that they are probably the same. Finally, we show that SpCCE1 can function as a Holliday junction resolvase in vivo by its ability to complement a resolvase-deficient strain of E. coli.

Effect of transverse compressive stress on the critical current and upper critical field of Nb3Sn

A large reversible degradation of the critical current of multifilamentary Nb3Sn superconductors has been observed when uniaxial compressive stress is applied transverse to the conductor axis at 4 K. In bronze-process multifilamentary Nb3Sn, the onset of significant degradation occurs at about 50 MPa. In an applied field of 10 T, the magnitude of the effect is about seven times larger for transverse stress than for stress applied along the conductor axis. The transverse stress effect increases with magnetic field and is associated with a reversible degradation of the upper critical field. The intrinsic effect of transverse stress on the upper critical field is about ten times greater than for axial stress. Although axial stresses on the Nb3Sn filaments are greater than transverse stresses in most applications, the transverse stress effect will need to be considered in the internal design of large magnets because of the greater sensitivity of Nb3Sn to transverse stress. It is shown that the transverse stress from the Lorentz force on the conductor is proportional to conductor thickness. This will place limits on conductor dimensions and the spacing between distributed reinforcement in large magnets. The effect may be particularly significant in cabled conductors where large transverse stress concentrations can occur at strand crossover points.

The topology of homologous pairing promoted by RecA protein

In addition to catalyzing the pairing of linear single-stranded DNA with homologous duplex DNA, recA protein promotes the pairing of circular single strands with linear duplex DNA or nicked circular duplex DNA, and of gapped circular duplex DNA with superhelical DNA. RecA protein will thus produce joint molecules of DNA at a high frequency from a pair of homologous molecules if one of them is single-stranded or partially single-stranded, and if either one has a free end. The structure made from a linear single strand and duplex DNA is a D loop. The joint molecule made from circular single-stranded DNA and linear duplex DNA is a branched structure in which the circular strand has displaced a strand from one end of the duplex molecule. In these structures, the heteroduplex regions reach sizes approaching that of full-length fd DNA. When we used restriction fragments of duplex fd DNA that were approximately half-length, we found circular molecules that were half duplex and half single-stranded. Similarly, single-stranded circles displaced a strand from nicked circular duplex DNA, yielding structures related to those made with linear duplex DNA, as well as other structures. Our observations indicate that purified recA protein catalyzes a concerted strand transfer with several features of particular biological interest, including the initiation of a strand crossover (in some cases perhaps the crossing back of a strand as well) and the production of long heteroduplex joints by a kind of branch migration. While a free end permits interwinding of DNA strands and the formation of joints containing stable right-handed helices, the free end is not essential for the promotion of homologous pairing by recA protein. When we mixed phage G4 double-stranded DNA and recA protein with single-stranded circular M13 DNA containing an insert of 274 bases of G4 DNA, we observed by electron microscopy the formation of a few percent of complexes in which single-stranded circular DNA and duplex DNA were joined side by side in the region of shared sequence homology. The frequency of such complexes was twenty to thirty times greater than that observed in a control mixture of G4 duplex DNA and single-stranded circular fd DNA, molecules which do not share a region of extensive homology. We conclude that recA protein can promote homologous association of a single strand and duplex DNA without the plectonemic colling that characterizes the normal Watson-Crick structure of DNA.