Single-stranded DNA Translocation Research Articles

Slowing the translocation of single-stranded DNA by using nano-cylindrical passage self-assembled by amphiphilic block copolymers

We report a novel approach to slow the translocation of single-stranded DNA (ssDNA) by employing polyethylene oxide (PEO) filled nano-cylindrical domains as transportation channels. DNA strands were demonstrated to electrophoretically translocate through PEO filled cylindrical domains with diameters of 2 and 9 nm, which were self-assembled by amphiphilic liquid crystalline block copolymers. The average translocation rate of ssDNA strands was effectively reduced to an order of 10 μs per nucleotide, which was 1-2 orders slower than that attained by utilizing conventional solid-state nanopore devices.

Intrinsic Stepwise Translocation of Stretched ssDNA in Graphene Nanopores

We investigate by means of molecular dynamics simulations stretch-induced stepwise translocation of single-stranded DNA (ssDNA) through graphene nanopores. The intrinsic stepwise DNA motion, found to be largely independent of size and shape of the graphene nanopore, is brought about through alternating conformational changes between spontaneous adhesion of DNA bases to the rim of the graphene nanopore and unbinding due to mechanical force or electric field. The adhesion reduces the DNA bases’ vertical conformational fluctuations, facilitating base detection and recognition. A graphene membrane shaped as a quantum point contact permits, by means of transverse electronic conductance measurement, detection of the stepwise translocation of the DNA as predicted through quantum mechanical Green’s function-based transport calculations. The measurement scheme described opens a route to enhance the signal-to-noise ratio not only by slowing down DNA translocation to provide sufficient time for base recognition but also by stabilizing single DNA bases and, thereby, reducing thermal noise.

Direct and Scalable Deposition of Atomically Thin Low-Noise MoS2 Membranes on Apertures.

Molybdenum disulfide (MoS2) flakes can grow beyond the edge of an underlying substrate into a planar freestanding crystal. When the substrate edge is in the form of an aperture, reagent-limited nucleation followed by edge growth facilitate direct and selective growth of freestanding MoS2 membranes. We have found conditions under which MoS2 grows preferentially across micrometer-scale prefabricated solid-state apertures in silicon nitride membranes, resulting in sealed membranes that are one to a few atomic layers thick. We have investigated the structure and purity of our membranes by a combination of atomic-resolution transmission electron microscopy, elemental analysis, Raman spectroscopy, photoluminescence spectroscopy, and low-noise ion-current recordings through nanopores fabricated in such membranes. Finally, we demonstrate the utility of fabricated ultrathin nanopores in such membranes for single-stranded DNA translocation detection.

The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling.

DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom’s syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

Replication and Active Partition of Integrative and Conjugative Elements (ICEs) of the SXT/R391 Family: The Line between ICEs and Conjugative Plasmids Is Getting Thinner.

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.

Surface Modification of Solid-State Nanopores for Sticky-Free Translocation of Single-Stranded DNA

Surface Modification of Solid-State Nanopores for Sticky-Free Translocation of Single-Stranded DNA

Mechanism of RecQ helicase mechanoenzymatic coupling reveals that the DNA interactions of the ADP-bound enzyme control translocation run terminations.

The processing of various DNA structures by RecQ helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Bloom's syndrome (BLM) helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning.

Surface modification of solid-state nanopores for sticky-free translocation of single-stranded DNA.

Nanopore technology is one of the most promising approaches for fast and low-cost DNA sequencing application. Single-stranded DNA detection is primary objective in such device, while solid-state nanopores remain less explored than their biological counterparts due to bio-molecule clogging issue caused by surface interaction between DNA and nanopore wall. By surface coating a layer of polyethylene glycol (PEG), solid-state nanopore can achieve long lifetime for single-stranded DNA sticky-free translocation at pH 11.5. Associated with elimination of non-specific binding of molecule, PEG coated nanopore presents new surface characteristic as less hydrophility, lower 1/f noise, and passivated surface charge responsiveness on pH. Meanwhile, conductance blockage of single-stranded DNA is found to be deeper than double-stranded DNA, which can be well described by a string of blobs model for a quasi-equilibrium state polymer in constraint space.

Slowing single-stranded DNA translocation through a solid-state nanopore by decreasing the nanopore diameter

To slow the translocation of single-stranded DNA (ssDNA) through a solid-state nanopore, a nanopore was narrowed, and the effect of the narrowing on the DNA translocation speed was investigated. In order to accurately measure the speed, long (5.3 kb) ssDNA (namely, ss-poly(dA)) with uniform length (±0.4 kb) was synthesized. The diameters of nanopores fabricated by a transmission electron microscope were controlled by atomic-layer deposition. Reducing the nanopore diameter from 4.5 to 2.3 nm slowed down the translocation of ssDNA by more than 16 times (to 0.18 μs base−1) when 300 mV was applied across the nanopore. It is speculated that the interaction between the nanopore and the ssDNA dominates the translocation speed. Unexpectedly, the translocation speed of ssDNA through the 4.5 nm nanopore is more than two orders of magnitude higher than that of double-stranded DNA (dsDNA) through a nanopore of almost the same size. The cause of such a faster translocation of ssDNA can be explained by the weaker drag force inside the nanopore. Moreover, the measured translocation speeds of ssDNA and dsDNA agree well with those calculated by molecular-dynamics (MD) simulation. The MD simulation predicted that reducing the nanopore diameter to almost the same as that of ssDNA (i.e. 1.4 nm) decreases the translocation speed (to 1.4 μs base−1). Narrowing the nanopore is thus an effective approach for accomplishing nanopore DNA sequencing.

Visualization of human Bloom's syndrome helicase molecules bound to homologous recombination intermediates

Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Bloom's syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM(642-1290)) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.

Direct imaging of single UvrD helicase dynamics on long single-stranded DNA

Fluorescence imaging of single-protein dynamics on DNA has been largely limited to double-stranded DNA or short single-stranded DNA. We have developed a hybrid approach for observing single proteins moving on laterally stretched kilobase-sized ssDNA. Here we probed the single-stranded DNA translocase activity of Escherichia coli UvrD by single fluorophore tracking, while monitoring DNA unwinding activity with optical tweezers to capture the entire sequence of protein binding, single-stranded DNA translocation and multiple pathways of unwinding initiation. The results directly demonstrate that the UvrD monomer is a highly processive single-stranded DNA translocase that is stopped by a double-stranded DNA, whereas two monomers are required to unwind DNA to a detectable degree. The single-stranded DNA translocation rate does not depend on the force applied and displays a remarkable homogeneity, whereas the unwinding rate shows significant heterogeneity. These findings demonstrate that UvrD assembly state regulates its DNA helicase activity with functional implications for its stepping mechanism, and also reveal a previously unappreciated complexity in the active species during unwinding.

Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase

Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3' to 5' translocase) and RecD (5' to 3' translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5' to 3' rate is always faster than the 3' to 5' rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3' to 5' and 5' to 3' translocation, whereas RecD only regulates 5' to 3' translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding.

Assessing graphene nanopores for sequencing DNA.

Using all-atom molecular dynamics and atomic-resolution Brownian dynamics, we simulate the translocation of single-stranded DNA through graphene nanopores and characterize the ionic current blockades produced by DNA nucleotides. We find that transport of single DNA strands through graphene nanopores may occur in single nucleotide steps. For certain pore geometries, hydrophobic interactions with the graphene membrane lead to a dramatic reduction in the conformational fluctuations of the nucleotides in the nanopores. Furthermore, we show that ionic current blockades produced by different DNA nucleotides are, in general, indicative of the nucleotide type, but very sensitive to the orientation of the nucleotides in the nanopore. Taken together, our simulations suggest that strand sequencing of DNA by measuring the ionic current blockades in graphene nanopores may be possible, given that the conformation of DNA nucleotides in the nanopore can be controlled through precise engineering of the nanopore surface.

How to Build a DNA Unwinding Machine

In this issue of Structure, He and colleagues present the crystal structure of the phage T4 Dda helicase bound to DNA, providing new insights into how this protein efficiently couples single-stranded DNA translocation to DNA strand separation.

Slowing down and stretching DNA with an electrically tunable nanopore in a p–n semiconductor membrane

We have studied single-stranded DNA translocation through a semiconductor membrane consisting of doped p and n layers of Si forming a p–n-junction. Using Brownian dynamics simulations of the biomolecule in the self-consistent membrane–electrolyte potential obtained from the Poisson–Nernst–Planck model, we show that while polymer length is extended more than when its motion is constricted only by the physical confinement of the nanopore. The biomolecule elongation is particularly dramatic on the n-side of the membrane where the lateral membrane electric field restricts (focuses) the biomolecule motion more than on the p-side. The latter effect makes our membrane a solid-state analog of the α-hemolysin biochannel. The results indicate that the tunable local electric field inside the membrane can effectively control dynamics of a DNA in the channel to either momentarily trap, slow down or allow the biomolecule to translocate at will.

Single-Stranded DNA Translocation of E. coli UvrD Monomer Is Tightly Coupled to ATP Hydrolysis

Escherichia coli UvrD is an SF1A (superfamily 1 type A) helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded DNA (ssDNA) translocase but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM), we proposed a nonuniform stepping mechanism in which a UvrD monomer translocates with biased (3′ to 5′) directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step size of 4–5 nt/step, suggesting that a pause occurs every 4–5 nt translocated. To further test this mechanism, we examined UvrD translocation over a range of lower ATP concentrations (10–500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ∼1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM), indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4–5 nt/step down to 50 μM ATP but increases to ∼7 nt/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP but with little futile ATP hydrolysis.

Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains

Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn2+-binding domain (residues 642–1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn2+-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein–protein interactions.

Dynamic Switch Between Assembly States of the Human Bloom's Syndrome Helicase during Homologous Recombination

During normal cellular functioning, various endogenous and exogenous DNA-damaging effects arise, including the production of reactive oxygen species and UV irradiation. Double-stranded DNA breaks (DSB) represent one of the most toxic forms of DNA damage, which can be lethal for the cell or induce malignant transformation. Homologous recombination (HR) is an essential and evolutionarily conserved pathway for the error-free repair of DSBs. However, excessive HR can also lead to harmful large-scale genome rearrangements. HR events are therefore tightly regulated. A key player in HR is the human Bloom's syndrome helicase (BLM). During HR, BLM exerts various molecular activities. Based on ensemble biochemical and single-molecule AFM studies we show that the different actions of BLM during HR take place in different oligomeric forms of the enzyme. During single-stranded DNA translocation, which serves as a basis for quality control of HR via disruption of Rad51 nucleoprotein filaments, BLM functions as a monomer. Contrary, more complex DNA structures resembling later HR intermediates, including D-loops and Holliday junctions, induce dimerization and higher-order oligomerization of BLM. The results indicate that BLM exists in a dynamic equilibrium between different assembly states, which is modulated by the structure of DNA intermediates encountered during HR.

Translocating Single-Stranded DNA through Crystalline Graphene Nanopores

Here, we report the successful translocation of single-stranded DNA through graphene nanopores which normally is plagued by sticking of the bases on the graphene, which we have now solved (we will reveal the details of this crucial issue at the BPS meeting). We recently developed a simple and fast water-based method for transferring graphene onto arbitrary surfaces, with micrometer alignment precision. Using this method, we fabricated ranges of graphene membranes in which nanometer sized pores were sculpted. Under an electron beam, we discovered that graphene undergoes a temperature sensitive self-repair mechanism that allows damage-free atomic scale sculpting of perfectly crystalline graphene nanostructures, such as nanopores with crystalline edges. When mounted between two flow chambers containing buffered DNA, these extremely thin nanopores were used to detect DNA molecules and very recently single-stranded strands of DNA. As individual DNA molecules translocate through the pore, characteristic temporary conductance changes were observed in the ionic current through the nanopore, setting the stage for future single-molecule genomic screening devices.

Translocation of single‐stranded DNA through the α‐hemolysin protein nanopore in acidic solutions

The effect of acidic pH on the translocation of single-stranded DNA through the α-hemolysin pore is investigated. Two significantly different types of events, i.e. deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, whereas the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to being used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing.