Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration.

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

Coarse-grained modelling of the structural properties of DNA origami.

We use the oxDNA coarse-grained model to provide a detailed characterization of the fundamental structural properties of DNA origami, focussing on archetypal 2D and 3D origami. The model reproduces well the characteristic pattern of helix bending in a 2D origami, showing that it stems from the intrinsic tendency of anti-parallel four-way junctions to splay apart, a tendency that is enhanced both by less screened electrostatic interactions and by increased thermal motion. We also compare to the structure of a 3D origami whose structure has been determined by cryo-electron microscopy. The oxDNA average structure has a root-mean-square deviation from the experimental structure of 8.4 Å, which is of the order of the experimental resolution. These results illustrate that the oxDNA model is capable of providing detailed and accurate insights into the structure of DNA origami, and has the potential to be used to routinely pre-screen putative origami designs and to investigate the molecular mechanisms that regulate the properties of DNA origami.

Molecular force spectroscopy with a DNA origami-based nanoscopic force clamp.

Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein-induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation.

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure.

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.

Top3α is required during the convergent migration step of double Holliday junction dissolution.

Although Blm and Top3α are known to form a minimal dissolvasome that can uniquely undo a double Holliday junction structure, the details of the mechanism remain unknown. It was originally suggested that Blm acts first to create a hemicatenane structure from branch migration of the junctions, followed by Top3α performing strand passage to decatenate the interlocking single strands. Recent evidence suggests that Top3α may also be important for assisting in the migration of the junctions. Using a mismatch-dHJ substrate (MM-DHJS) and eukaryotic Top1 (in place of Top3α), we show that the presence of a topoisomerase is required for Blm to substantially migrate a topologically constrained Holliday junction. When investigated by electron microscopy, these migrated structures did not resemble a hemicatenane. However, when Blm is together with Top3α, the dissolution reaction is processive with no pausing at a partially migrated structure. Potential mechanisms are discussed.

Precise structure control of three-state nanomechanical DNA origami devices.

Precise structure switching between all of the three forms of three-state nanomechanical DNA origami devices has been accomplished. A nanomechanical DNA origami device called DNA origami pliers, which consists of two levers of 170-nm long, 20-nm wide, and 2-nm thick connected at a Holliday-junction fulcrum, takes three conformations: closed parallel, closed antiparallel, and open cross forms. They were previously applied to construct detection systems for biomolecules in single-molecular resolution by observing the structure switching between cross form and one of the other two forms under atomic force microscope (AFM). We redesigned DNA origami pliers in this study to let them freely switch between all of the three states including parallel-antiparallel direct switching without taking cross form. By the addition of appropriate switcher strands to the solution, hybridization and dehybridization of particular binder strands that fix the levers into predetermined state were selectively triggered as programmed in their sequence. Circuit structure switching through all of the three states in both of the two opposite direction was even successful with the new design.

Improved methods for creating migratable Holliday junction substrates.

Previously, we published a method for creating a novel DNA substrate, the double Holliday junction substrate. This substrate contains two Holliday junctions that are mobile, topologically constrained and separated by a distance comparable with conversion tract lengths. Although useful for studying late stage homologous recombination in vitro, construction of the substrate requires significant effort. In particular, there are three bottlenecks: (i) production of large quantities of single-stranded DNA; (ii) the loss of a significant portion of the DNA following the recombination step; and (iii) the loss of DNA owing to inefficient gel extraction. To address these limitations, we have made the following changes to the protocol: (i) use of a helper plasmid, rather than exogenous helper phage, to produce single-stranded DNA; (ii) use of the unidirectional C31 integrase system in place of the bidirectional Cre recombinase reaction; and (iii) gel extraction by DNA diffusion. Here, we describe the changes made to the materials and methods and characterize the substrates that can be produced, including migratable single Holliday junctions, hemicatenanes and a quadruple Holliday junction substrate.

Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy.

In mammals, there are five Rad51 paralogs that form two distinct complexes in vivo. One complex is composed of Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and the other Rad51C-Xrcc3 (CX3). We co-expressed and purified human BCDX2 and CX3 protein complexes from insect cells and investigated their binding preferences and structure using transmission electron microscopy (TEM). We visualized the binding of BCDX2 and CX3 to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both complexes bind with exceptionally high specificity to the DNA junctions with little binding observed elsewhere on the DNAs. Further analysis of the structure of free or DNA-bound BCDX2 and CX3 complexes revealed a multimeric ring structure whose subunits are arranged into a flat disc around a central channel. This work provides the first EM visualization of BCDX2 and CX3 binding to Holliday junctions and forked DNAs and suggests the complexes form ring-shaped structures.

RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination.

Saccharomyces cerevisiae RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, are implicated in processing joint molecule (JM) recombination intermediates. We show that cells lacking either enzyme frequently experience chromosome segregation problems during meiosis and that when both enzymes are absent attempted segregation fails catastrophically. In all cases, segregation appears to be impeded by unresolved JMs. Analysis of the DNA events of recombination indicates that Sgs1 limits aberrant JM structures that result from secondary strand-invasion events and often require Mus81-Mms4 for their normal resolution. Aberrant JMs contain high levels of single Holliday junctions and include intersister JMs, multichromatid JMs comprising three and four chromatids, and newly identified recombinant JMs containing two chromatids, one of which has undergone crossing over. Despite persistent JMs in sgs1 mms4 double mutants, crossover and noncrossover products still form at high levels. We conclude that Sgs1 and Mus81-Mms4 collaborate to eliminate aberrant JMs, whereas as-yet-unidentified enzymes process normal JMs.

The Werner syndrome protein binds replication fork and holliday junction DNAs as an oligomer.

Werner syndrome is an inherited disease displaying a premature aging phenotype. The gene mutated in Werner syndrome encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Both WRN helicase and exonuclease preferentially utilize DNA substrates containing alternate secondary structures. By virtue of its ability to resolve such DNA structures, WRN is postulated to prevent the stalling and collapse of replication forks that encounter damaged DNA. Using electron microscopy, we visualized the binding of full-length WRN to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both wild-type WRN and a helicase-defective mutant bind with exceptionally high specificity (>1000-fold) to DNA secondary structures at the replication fork and at Holliday junctions. Little or no binding is observed elsewhere on the DNA molecules. Calculations of the molecular weight of full-length WRN revealed that, in solution, WRN exists predominantly as a dimer. However, WRN bound to DNA is larger; the mass is consistent with that of a tetramer.

Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex.

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.

DNA microstructure based on self-assembly of 4-sticky-end Holliday junctions in aqueous solution.

Liverwort-like DNA microscale structures consist of 4-sticky-end Holiday junctions as DNA bricks that can be used in nanotechnology and nanobiotechnology to direct the self-assembly of nanomachines as well as DNA assembly. Previously it has not been possible to obtain such DNA microscale structural forms, but herein we report construction of a mesh-like material made up of 4 strands of 40-base DNA. Advanced bioimaging techniques such as fluorescence correlation spectroscopy (FCS), laser scanning microscopy (LSM), and atomic force microscopy (AFM) help us as ultrasensitive detection tools for examing structures in solutions. Combinations of these techniques allow us to survey various chemical conditions of materials and solutions.

The basic domain of TRF2 directs binding to DNA junctions irrespective of the presence of TTAGGG repeats.

The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Ozgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and four-way junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2(DeltaB)) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junction-specific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop.

Direct observation of RuvAB-catalyzed branch migration of single Holliday junctions.

Holliday junctions form during DNA repair and homologous recombination processes. These processes entail branch migration, whereby the length of two arms of a cruciform increases at the expense of the two others. Branch migration is carried out in prokaryotic cells by the RuvAB motor complex. We study RuvAB-catalyzed branch migration by following the motion of a small paramagnetic bead tethered to a surface by two opposing arms of a single cruciform. The bead, pulled under the action of magnetic tweezers, exerts tension on the cruciform, which in turn transmits the force to a single RuvAB complex bound at the crossover point. This setup provides a unique means of measuring several kinetic parameters of interest such as the translocation rate, the processivity, and the force on the substrate against which the RuvAB complex cannot effect translocation. RuvAB-catalyzed branch migration proceeds with a small, discrete number of rates, supporting the view that the monomers comprising the RuvB hexameric rings are not functionally homogeneous and that dimers or trimers constitute the active subunits. The most frequently encountered rate, 98 +/- 3 bp/sec, is approximately five times faster than previously estimated. The apparent processivity of branch migration between pauses of inactivity is approximately 7,000 bp. Branch migration persists against opposing forces up to 23 pN.

Single-molecule study of RuvAB-mediated Holliday-junction migration.

Branch migration of Holliday junctions is an important step of genetic recombination and DNA repair. In Escherichia coli, this process is driven by the RuvAB complex acting as a molecular motor. Using magnetic tweezers, we studied the RuvAB-directed migration of individual Holliday junctions formed between two approximately 6-kb DNA molecules of identical sequence, and we measured the migration rate at 37 degrees C and 1 mM ATP. We directly demonstrate that RuvAB is a highly processive DNA motor protein that is able to drive continuous and unidirectional branch migration of Holliday junctions at a well defined average speed over several kilobases through homologous sequences. We observed directional inversions of the migration at the DNA molecule boundaries leading to forth-and-back migration of the branch point and allowing us to measure the migration rate in the presence of negative or positive loads. The average migration rate at zero load was found to be approximately 43 bp/sec. Furthermore, the load dependence of the migration rate is small, within the force range of -3.4 pN (hindering force) to +3.4 pN (assisting force).

Telomere maintenance requires the RAD51D recombination/repair protein.

The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d(-/-) Trp53(-/-) primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53(-/-) or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion.