Branch Migration Process Research Articles

Single-Molecule Analysis of DNA Branch Migration under Biomimetic Environments.

Branch migration (BM) of DNA Holliday junctions (HJs) occurs through base-pair rearrangements between homologous DNA molecules during the repair of double-strand breaks (DSBs) by homologous recombination. Despite the fact that BM is conserved among organisms and is essential to stabilize recombination intermediates, which occurs by avoiding the reversal of strand exchange leading to the faithful repair of DSBs, molecular insights into the BM process including kinetics, the effects of microenvironments, and the role of HJ-binding proteins are poorly understood. In this article, using single-molecule fluorescence analysis of a synthetic, mobile HJ as a model system, we systematically investigated the effects of cell-mimic solvent composition and crowding on BM by varying the concentration of cosolutes, namely dimethyl sulfoxide (DMSO) and poly(ethylene glycol) (PEG). The single-molecule analyses revealed that BM is affected by cosolutes in a concentration-dependent manner. In addition, the kinetic analysis of the fluorescence resonance energy transfer (FRET) traces showed that the BM is significantly accelerated under the crowding environments. The finding that the mobility of the HJ is enhanced by cell-like environments not only provides a better insight into BM but also may open up new avenues for targeting HJs and the associated BM process for possible therapeutic interventions.

Elucidation of Interactions Between Integration Host Factor and A DNA Four‐Way Junction

Integration host factor (IHF) is an E. coli nucleoid-associated protein that was initially discovered in the integration of bacterial phage DNA. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence and induces a bend of ~160° upon binding. Our previous results have shown that IHF binds to DNA 4WJ that do not contain the consensus sequence with high affinity. DNA Four-Way Junctions (4WJ) are important intermediates in double-strand break repair and homologous recombination. These junctions can adopt either an open or stacked conformation, in which the open conformation facilitates the process of branch migration and strand exchange. In this study, we investigated the IHF-induced changes in the population distribution of junction conformations, using ensemble and single molecule Förster resonance energy transfer (FRET) methods. Our steady state and time-resolved FRET data indicated that under conditions stabilizing the stacked junction conformation, upon binding, IHF induces the open conformation of junction. Single-molecule FRET (smFRET) experiments, performed with total internal reflection fluorescence microscopy, measured population distributions of the junction and showed clear opening of the junctions upon IHF binding. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Cross correlation analyses reveal that the protein slows down the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+, consistent with stabilization of the open conformation. These findings which show that IHF binds to 4WJ with high affinity, induces the open conformation and alters conformational dynamics, suggest that IHF may play multiple roles in the processes of integration and recombination.

Biochemical Logic Circuits Based on DNA Combinatorial Displacement

DNA, as an excellent nano-engineering material, contributes to a new computing model, namely, DNA computing. This model is a type of biological computing, which takes advantage of the high density and high parallelism of molecules. One of the current methods of implementing DNA computing is to construct DNA circuits, among which the toehold-mediated DNA strand displacement technique is an important method. The hybridization of toehold domains provides the start position and accelerates the branch migration process. Toehold-based DNA combinatorial displacement is a practical method for designing and implementing DNA circuits. In this paper, we designed and simulated a multiplexer using the DNA combinatorial displacement mechanism to verify its practicability. Additionally, we improved and optimized the existing logic INHIBIT gate by leveraging the DNA combinatorial displacement mechanism so that the DNA strands in the entire chemical reaction network (CRN) system are capable of coexisting in large quantities. Moreover, we applied this improvement to the demultiplexer. Our method provides more capabilities to larger and more complicated DNA integrated circuits.

Modulating Responses of Toehold Switches by an Inhibitory Hairpin.

The toehold switch consists of a cis-repressing switch RNA hairpin and a trans-acting trigger RNA. The binding of the trigger RNA to an unpaired toehold sequence of the switch hairpin allows for a branch migration process, exposing the start codon and ribosome binding site for translation initiation. In this work, we demonstrate that responses of toehold switches can be modulated by introducing an inhibitory hairpin that shortens the length of the unpaired toehold region. First, we investigated the effect of the toehold region length on output gene expression and showed that the second trigger RNA, which binds to the inhibitory hairpin, is necessary for output gene activation when the hairpin-to-hairpin spacing is short. Second, the apparent Hill coefficient was found generally to increase with decreasing hairpin-to-hairpin spacing or increasing hairpin number. This work expands the utility of toehold switches by providing a new way to modulate their response.

Measuring and Modeling the Effect of Single Mismatch on DNA Strand Displacement

DNA strand displacement is an essential reaction in both DNA homologous recombination and DNA mismatch repair, and it is also heavily utilized in DNA-based computation and locomotion. Despite this ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Furthermore, the bulk methods currently used make it difficult to study the underlying branch migration process. Here, we directly measured the branch migration kinetics of toehold-mediated strand displacement using single-molecule fluorescence in the presence of a single nucleotide mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data reveal direct dissociation of the shorter DNA strand (incumbent) due to weakened base pairing during branch migration as the dominant pathway for DNA strand displacement. We show that a simple first passage model can quantitatively explain the salient features of the observed relationship. We expect our model to become a powerful tool to design DNA-based reaction schemes with broad functionality.

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21pM, providing a simple, sensitive and selective protocol for small target molecules detection.

A G-quadruplex DNAzyme chemiluminescence aptasensor based on the target triggered DNA recycling for sensitive detection of adenosine

A G-quadruplex DNAzyme chemiluminescence aptasensor based on the target triggered DNA recycling was constructed for sensitive detection of adenosine. The system involves four nucleic acid strands, namely DNA1, DNA2, H1 and H2. DNA1 is a chimeric conjugate of the adenosine aptamer strand, DNA2 is an inhibitory strand that contains complementary sequences to DNA1. Hairpin structures H1 and H2 are designed according to the sequences of DNA2. In the absence of adenosine, DNA1 is inhibited by hybridizing with DNA2, and H1 and H2 coexist in solution. In the presence of adenosine, the DNA1 strand binds adenosine, and results in the release of DNA2. Upon interaction of DNA2 with H1, the hairpin structure of H1 is opened, resulting in the activation of the G-quadruplex. Meanwhile, the unfolded region in H1 can serve as a toehold to hybridize with H2, leading to the displacement of the DNA2 through a branch migration process. Thus, the DNA2 dissociates from the H1–H2 complex. The released DNA2 hybridizes with another H1 and initiates the second cycle, and generates a number of H1–H2 complexes containing the G-riched DNA. The G-riched DNA assembles with hemin to form the hemin–G-quadruplexes that exhibit peroxidase-like activity which can catalyze oxidation of luminol by H2O2 generating chemiluminescence. In this way, a single adenosine can trigger many H1–H2 complexes and activate many G-quadruplex DNAzymes, which leads to the significant chemiluminescence enhancement. The linear detection range of adenosine with the proposed aptasensor is 0.8 to 200 μM, and the detection limit comes down to 0.6 μM. Most importantly, no organic dye molecule-labeled DNA or extra nanomaterials were used in this proposed CL aptasensor. The method is simple, sensitive and inexpensive and has potential for application in clinical diagnostic assays.

Internal Friction of a Migrating Holliday Junction

Friction within biomolecules has recently gained increasing interest. Here we present a method that allows to study the friction that occurs during fast, few nm-sized refolding processes of nucleic acids. Branch migration of a homologous Holliday junction serves as an ideal system where such friction can be investigated. In this four-arm DNA junction the opposing arms possess identical sequences with respect to the junction center. In the absence of external constraints the junction is mobile such that one pair of homologous arms can expand at the expense of the other in single base pair diffusive steps. We measure the dynamics of the branch migration process by stretching a torsionally constrained Holliday junction using magnetic tweezers and measuring the length fluctuations of the arms with high-speed videomicroscopy at ∼3 kHz. Since DNA has a helical structure, branch migration causes twisting of the arms with one turn per helical pitch moved. This constrains the movement of the junction within the tweezers to ∼10 bp. Single base pair diffusive steps are expected to occur on a sub-millisecond time scale and to be much smaller than the overall DNA length fluctuations. Thus they cannot be directly resolved. However, power-spectral-density analysis of the length fluctuations is able to clearly resolve the overall dynamics of the branch migration process. Theoretical modeling considering the elastic coupling of DNA bending fluctuations and the junction movement allows to quantitatively determine the stepping rate and thus the friction of the branch migration process. We expect that our method is widely applicable to study local-scale molecular friction in biological systems.

Hop2-Mnd1 Condenses DNA to Stimulate the Synapsis Phase of DNA Strand Exchange

Hop2-Mnd1 is a meiotic recombination mediator that stimulates DNA strand invasion by both Dmc1 and Rad51. To understand the biochemical mechanism of this stimulation, we directly visualized the heterodimer acting on single molecules of duplex DNA using optical tweezers and video fluorescence microscopy. The results show that the Hop2-Mnd1 heterodimer efficiently condenses double-stranded DNA via formation of a bright spot or DNA condensate. The condensation of DNA is Hop2-Mnd1 concentration-dependent, reversible, and specific to the heterodimer, as neither Hop2 nor Mnd1 acting alone can facilitate this reaction. The results also show that the rate-limiting nucleation step of DNA condensation is overcome in the presence of divalent metal ions, with the following order of preference: Mn2+>Mg2+>Ca2+. Hop2-Mnd1/Dmc1/single-stranded DNA nucleoprotein filaments also condense double-stranded DNA in a heterodimer concentration-dependent manner. Of importance, the concentration dependence parallels that seen in DNA strand exchange. We propose that rapid DNA condensation is a key factor in stimulating synapsis, whereas decondensation may facilitate the invasion step and/or the ensuing branch migration process.

Single Molecule Fluorescence Analysis of Branch Migration of Holliday Junctions: Effect of DNA Sequence

The Holliday junction is a central intermediate in various genetic processes including homologous, site-specific recombination and DNA replication. Recent single molecule FRET experiments led to the model for branch migration as a stepwise stochastic process in which the branch migration hop is terminated by the folding of the junction. In this article, we studied the effect of the sequence on Holliday junction dynamics and branch migration process. We show that a GC pair placed at the border of the homologous region almost prevents the migration into this position. At the same time, insertion of a GC pair into the middle of the AT tract does not show this effect, however when the junction folds at this position, it resides at this position much longer time in comparison to the folding at AT pairs. Two contiguous GC pairs do not block migration as well and generally manifest the same effect as one GC pair—the junction when it folds resides at these positions for a relatively long time. The same elevated residence time was obtained for the design with the homology region that consists of only GC pairs. These data suggest a model for branch migration in which the sequence modulates the overall stochastic process of the junction dynamics and branch migration by the variability of the time that the junction dwells before making a migration hop.

Branch migration displacement assay with automated heuristic analysis for discrete DNA length measurement using DNA microarrays

The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics and biology. Here we demonstrate a rapid, highly sensitive method for STR analysis that utilizes DNA microarray technology. We describe two adaptations to accomplish this: the use of competitive hybridization to remove unpaired ssDNA from an array and the use of neural network classification to automate the analysis. The competitive displacement technique mimics the branch migration process that occurs during DNA recombination. Our technique will facilitate the rapid deduction of identity, length, and number of repeats for the multiple STRs in an unknown DNA sample.

Selective Binding of Meiosis-specific Yeast Hop1 Protein to the Holliday Junctions Distorts the DNA Structure and Its Implications for Junction Migration and Resolution

Saccharomyces cerevisiae HOP1, which encodes a component of synaptonemal complex (SC), plays an important role in both gene conversion and crossing over between homologs, as well as enforces meiotic recombination checkpoint control over the progression of recombination intermediates. In hop1Δ mutants, meiosis-specific double-strand breaks (DSBs) are reduced to10% of the wild-type level, and at aberrantly late times, these DSBs are processed into inter-sister recombination intermediates. However, the underlying mechanism by which Hop1 protein regulates these nuclear events remains obscure. Here we show that Hop1 protein interacts selectively with the Holliday junction, changes its global conformation and blocks the dissolution of the junction by a RecQ helicase. The Holliday junction–Hop1 protein complexes are significantly more stable at higher ionic strengths and molar excess of unlabeled competitor DNA than complexes containing other recombination intermediates. Structural analysis of the Holliday junction using 2-aminopurine fluorescence emission, DNase I footprinting and KMnO 4 probing provide compelling evidence that Hop1 protein binding induces significant distortion at the center of the Holliday junction. We propose that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates with the process of branch migration of Holliday junction.

Using DNA to construct and power a nanoactuator.

A DNA-based molecular machine is described which has two movable arms that are pushed apart when a strand of DNA, the fuel strand, hybridizes with a single-stranded region of the molecular machine. Through the process of branch migration, a second strand of DNA complementary to the fuel strand is able to remove the fuel strand from the molecular machine, restoring it to its original configuration. Compared with the molecular tweezers we had previously devised, this machine, which we call a nanoactuator, has a reduced tendency to form dimers.

DNA hybrids stabilized by heterologies.

The double D-loop DNA hybrid contains four DNA strands following hybridization of two RecA protein coated complementary single-stranded DNA probes with a homologous region of a double-stranded DNA target. A remarkable feature of the double D-loop DNA hybrids is their kinetic stabilities at internal sites within linear DNA targets after removal of RecA protein from hybrids. We report here that heterologous DNA inserts in one or both probe strands affect the kinetic stability of protein-free double D-loop hybrids. DNA heterologies normally distort DNA-DNA hybrids and consequently accelerate hybrid dissociation. In contrast, heterologous DNA inserts impede dissociation of double D-loops, especially when the insert sequences interact with each other by DNA base pairing. We propose a mechanism for this kinetic stabilization by heterologous DNA inserts based on the hypothesis that the main pathway of dissociation of double D-loop DNA hybrids is a DNA branch migration process involving the rotation of both probe-target duplexes in the hybrids. Heterologous DNA inserts constrain rotation of probe-target duplexes and consequently impede hybrid dissociation. Potential applications of the stabilized double D-loops for gene targeting are discussed.

DNA cruciforms facilitate in vitro strand transfer on nucleosomal templates.

A single, phased nucleosome assembled on a 240 bp DNA duplex molecule blocked Escherichia coli RecA protein-promoted strand transfer of the complementary strand of the duplex onto a homologous single-stranded circle. However, when a four-armed cruciform structure was coupled to either end of the duplex the barrier to strand transfer was overcome and joint molecules were efficiently formed. Micrococcal nuclease digestion indicated that the nucleosome was dissociated by the juxtaposition of the cruciform. We interpret these results to mean that cruciform structures can act over a distance to destabilize adjacent nucleosomes and suggest that, as a consequence, the chromatin structure surrounding a crossed strand recombination intermediate might be disrupted, enabling other recombination events to initiate or the process of branch migration to proceed.

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.

A model for replication repair in mammalian cells.

Abstract A model for replication repair based on the process of branch migration explains the transient production of doubly substituted DNA within the first generation of incubation in bromodeoxyuridine and the appearance of four-pronged replication forks.