Structural insights into the mechanism of DNA branch migration during homologous recombination in bacteria.

Some DNA helicases play central and specific roles in genome maintenance and plasticity through their branch migration activity in different pathways of homologous recombination. RadA is a highly conserved bacterial helicase involved in DNA repair throughout all bacterial species. In Gram-positive Firmicutes, it also has a role in natural transformation, while in Gram-negative bacteria, ComM is the canonical transformation-specific helicase. Both RadA and ComM helicases form hexameric rings and use ATP hydrolysis as an energy source to propel themselves along DNA. In this study, we present the cryoEM structures of RadA and ComM interacting with DNA and ATP analogs. These structures reveal important molecular interactions that couple ATP hydrolysis and DNA binding in RadA, as well as the role of the Lon protease-like domain, shared by RadA and ComM, in this process. Taken together, these results provide new molecular insights into the mechanisms of DNA branch migration in different pathways of homologous recombination.

Viral Genomic DNA Packaging Machinery.

Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

Over 30-Fold Enhancement in DNA Translocation Dynamics through Nanoscale Pores Coated with an Anionic Surfactant.

Solid-state nanopores (ssNPs) are single-molecule sensors capable of label-free quantification of different biomolecules, which have become highly versatile with the introduction of different surface treatments. By modulating the surface charges of the ssNP, the electro-osmotic flow (EOF) can be controlled in turn affecting the in-pore hydrodynamic forces. Herein, we demonstrate that negative charge surfactant coating to ssNPs generates EOF that slows-down DNA translocation speed by >30-fold, without deterioration of the NP noise, hence significantly improving its performances. Consequently, surfactant-coated ssNPs can be used to reliably sense short DNA fragments at high voltage bias. To shed light on the EOF phenomena inside planar ssNPs, we introduce visualization of the electrically neutral fluorescent molecule's flow, hence decoupling the electrophoretic from EOF forces. Finite elements simulations are then used to show that EOF is likely responsible for in-pore drag and size-selective capture rate. This study broadens ssNPs use for multianalyte sensing in a single device.

Uncovering a New Step in Sliding Nucleosomes.

Chromatin remodelers are ATP-driven motors that pump double-stranded DNA around the histone core of the nucleosome. Recent work by Chen and coworkers (Li et al., Nature, 2019 and Yan et al., Nat. Struct. Mol. Biol., 2019) has revealed an unexpected intermediate where initial translocation involves only one of the two DNA strands.

FtsK in motion reveals its mechanism for double-stranded DNA translocation.

FtsK protein contains a fast DNA motor that is involved in bacterial chromosome dimer resolution. During cell division, FtsK translocates double-stranded DNA until both dif recombination sites are placed at mid cell for subsequent dimer resolution. Here, we solved the 3.6-Å resolution electron cryo-microscopy structure of the motor domain of FtsK while translocating on its DNA substrate. Each subunit of the homo-hexameric ring adopts a unique conformation and one of three nucleotide states. Two DNA-binding loops within four subunits form a pair of spiral staircases within the ring, interacting with the two DNA strands. This suggests that simultaneous conformational changes in all ATPase domains at each catalytic step generate movement through a mechanism related to filament treadmilling. While the ring is only rotating around the DNA slowly, it is instead the conformational states that rotate around the ring as the DNA substrate is pushed through.

Ultrafast Polymer Dynamics through a Nanopore.

Ultrathin nanopore sensors allow single-molecule and polymer measurements at sub-microsecond time resolution enabled by high current signals (∼10-30 nA). We demonstrate for the first time the experimental probing of the ultrafast translocation and folded dynamics of double-stranded DNA (dsDNA) through a nanopore at 10 MHz bandwidth with acquisition of data points per 25 ns (150 MB/s). By introducing a rigorous algorithm, we are able to accurately identify each current level present within translocation events and elucidate the dynamic folded and unfolded behaviors. The remarkable sensitivity of this system reveals distortions of short-lived folded states at a lower bandwidth. This work revisits probing of dsDNA as a model polymer and develops broadly applicable methods. The combined improvements in sensor signals, instrumentation, and large data analysis methods uncover biomolecular dynamics at unprecedentedly small time scales.

Single Molecule DNA Analysis Based on Atomic-Controllable Nanopores in Covalent Organic Frameworks.

We explored the application of two-dimensional covalent organic frameworks (2D COFs) in single molecule DNA analysis. Two ultrathin COF nanosheets were exfoliated with pore sizes of 1.1 nm (COF-1.1) and 1.3 nm (COF-1.3) and covered closely on a quartz nanopipette with an orifice of 20 ± 5 nm. COF nanopores exhibited high size selectivity for fluorescent dyes and DNA molecules. The transport of long (calf thymus DNA) and short (DNA-80) DNA molecules through the COF nanopores was studied. Because of the strong interaction between DNA bases and the organic backbones of COFs, the DNA-80 was transported through the COF-1.1 nanopore at a speed of 270 µs/base, which is the slowest speed ever observed compared with 2D inorganic nanomaterials. This study shows that the COF nanosheet can work individually as a nanopore monomer with controllable pore size like its biological counterparts.

Modifying surface charge density of thermoplastic nanofluidic biosensors by multivalent cations within the slip plane of the electric double layer.

Thermoplastic nanofluidic devices are promising platforms for sensing single biomolecules due to their mass fabrication capability. When the molecules are driven electrokinetically through nanofluidic networks, surface charges play a significant role in the molecular capture and transportation, especially when the thickness of the electrical double layer is close to the dimensions of the nanostructures in the device. Here, we used multivalent cations to alter the surface charge density of thermoplastic nanofluidic devices. The surface charge alteration was done by filling the device with a multivalent ionic solution, followed by withdrawal of the solution and replacing it with KCl for conductance measurement. A systematic study was performed using ionic solutions containing Mg2+ and Al3+ for nanochannels made of three polymers: poly(ethylene glycol) diacrylate (PEGDA), poly(methyl methacrylate) (PMMA) and cyclic olefin copolymer (COC). Overall, multivalent cations within the slip plane decreased the effective surface charge density of the device surface and the reduction rate increased with the cation valency, cation concentration and the surface charge density of thermoplastic substrates. We demonstrated that a 10-nm diameter in-plane nanopore formed in COC allowed translocation of λ-DNA molecules after Al3+ modification, which is attributed to the deceased viscous drag force in the nanopore by the decreased surface charge density. This work provides a general method to manipulate surface charge density of nanofluidic devices for biomolecule resistive pulse sensing. Additionally, the experimental results support ion-ion correlations as the origin of charge inversion over specific chemical adsorption.

DNA translocation through pH-dependent soft nanopores.

Controlling the translocation velocity of DNA is the main challenge in the process of sequencing by means of nanopores. One of the main methods to overcome this challenge is covering the inner walls of the nanopore with a layer of polyelectrolytes, i.e., using soft nanopores. In this paper the translocation of DNA through soft nanopores, whose inner polyelectrolyte layer (PEL) charge is pH-dependent, is theoretically studied. We considered the polyelectrolyte to be made up of either acidic or basic functional groups. It was observed that the electroosmotic flow (EOF) induced by the PEL charge is in the opposite/same direction of DNA electrophoresis (EPH) when the PEL is made up of acidic/basic groups. It was found that, not only the DNA charge and consequently the EPH, but also the EOF are influenced by the electrolyte acidity. The synergy between the changes in the retardation, EOF and EPH, determines how the intensity and direction of DNA translocation alter with pH. In fact, for both cases, at mild values of pH (as long as [Formula: see text] for the case that PEL is of acidic nature), the more the pH, the less the translocation velocity. However, for PELs of acidic nature, higher values of pH increase the intensity of the EOF so much that DNA may experience a change in the translocation direction. Ultimately, conducting the process at a particular range of pH values, and at higher pH values, in the cases of using PELs of acidic nature, and basic nature, respectively, was recommended.

Deciphering the structure of the condensin protein complex.

Protein assemblies consisting of structural maintenance of chromosomes (SMC) and kleisin subunits are essential for the process of chromosome segregation across all domains of life. Prokaryotic condensin belonging to this class of protein complexes is composed of a homodimer of SMC that associates with a kleisin protein subunit called ScpA. While limited structural data exist for the proteins that comprise the (SMC)-kleisin complex, the complete structure of the entire complex remains unknown. Using an integrative approach combining both crystallographic data and coevolutionary information, we predict an atomic-scale structure of the whole condensin complex, which our results indicate being composed of a single ring. Coupling coevolutionary information with molecular-dynamics simulations, we study the interaction surfaces between the subunits and examine the plausibility of alternative stoichiometries of the complex. Our analysis also reveals several additional configurational states of the condensin hinge domain and the SMC-kleisin interaction domains, which are likely involved with the functional opening and closing of the condensin ring. This study provides the foundation for future investigations of the structure-function relationship of the various SMC-kleisin protein complexes at atomic resolution.

The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies.

TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation.

Increased dwell time and occurrence of dsDNA translocation events through solid state nanopores by LiCl concentration gradients.

Solid-state nanopore based biosensors are cost effective, high-throughput engines for single molecule detection of biomolecules with the added benefit of size modification. Progress in the translation of the science into a viable diagnostic tool is impeded by inadequate sensitivity of data acquisition systems in detection of fast DNA translocations through the pore. To combat this, slowing the transport of DNA through the nanopore by use of various media or by altering experimental parameters is common. Applying a concentration gradient of KCl in the experimental ionic solution has been shown to effectively prolong dwell times as well as increase the capture rate of DNA by the nanopore. Our previous work has corroborated the ability of LiCl ionic solution to slow down the transport of dsDNA through the nanopore by up to 10-fold through cation-DNA interactions. However, this drastically reduced the event occurrence frequency, thus hindering the efficacy of this system as a reliable biosensor downstream. Here, we present the use of a concentration gradient of lithium chloride ionic solution to increase the event frequency of single molecule dsDNA translocation through a solid state nanopore. By using 0.5 M/3 M LiCl on the cis/trans chambers respectively, average dwell times experienced up to a 3-fold increase when compared to experiments run in symmetric 1 M LiCl. Additionally, experiments using the 0.5 M/3 M displayed a greater than 10-fold increase in event frequency, confirming the capture propensity of the asymmetric conditions.

Orthogonal Tip-to-Tip Nanocapillary Alignment Allows for Easy Detection of Fluorescent Emitters in Femtomolar Concentrations.

Here we present the realization of a novel fluorescence detection method based on the electromigration of fluorescent molecules within a nanocapillary combined with the laser excitation through a platinum (Pt)-coated nanocapillary. By using the Pt nanocapillary assisted focusing of a laser beam, we completely remove the background scattering on the tip of the electrophoretic nanocapillary. In this excitation geometry, we demonstrate a 1000-fold sensitivity enhancement (1.0 nM to 1.0 pM) compared to the detection in microcapillaries with epifluorescence illumination and fluorescence spectrophotometry. Due to a significant electroosmotic flow, we observe a decelerating migration of DNA molecules close to the tip of the electrophoretic nanocapillary. The reduced DNA translocation velocity causes a two-step stacking process of molecules in the tip of the nanocapillary and can be used as a way to locally concentrate molecules. The sensitivity of our method is further improved by a continuous electrokinetic injection of DNA molecules followed by sample zone stacking on the tip of the nanocapillary. Concentrations ranging from 0.1 pM to 1.0 fM can be directly observed on the orifice of the electrophoretic nanocapillary. This is a 1000-fold improvement compared to traditional capillary electrophoresis with laser-induced fluorescence.

Solid-state nanopore fabrication in LiCl by controlled dielectric breakdown.

Nanopore fabrication via the controlled dielectric breakdown (CDB) method offers an opportunity to create solid-state nanopores directly in salt solution with sub-nanometer precision. Driven by trap assisted current tunneling, the method uses localized defects, or traps, in the dielectric material to isolate a breakdown point and fabricate a single pore in less than 10 minutes. Here we present an approach to controlled dielectric breakdown of SiNx in which the nanopore is fabricated in LiCl buffer instead of the traditional KCl buffer. Direct fabrication in LiCl buffer promotes a uniform, symmetric, cylindrical nanopore structure that is fully wet and can be used for experiments in situ. We have shown that fabrication in LiCl reduces the necessity for overnight pore stabilization and allows for the desired analyte to be added in significantly less time than it would take if fabrication was performed in KCl. Pores created by this approach can be used for biosensing applications, including the detection of double-stranded DNA. DNA translocation experiments were conducted in both LiCl and KCl buffer. Experiments conducted in LiCl buffer resulted in about a 2-fold increase in dsDNA transport duration when compared to experiments conducted in KCl buffer of the same concentration. An increase in transport duration of over 10-fold in comparison to KCl was observed when the concentration of the LiCl buffer was increased by a factor of 3.

Assembly, translocation, and activation of XerCD-dif recombination by FtsK translocase analyzed in real-time by FRET and two-color tethered fluorophore motion.

The FtsK dsDNA translocase functions in bacterial chromosome unlinking by activating XerCD-dif recombination in the replication terminus region. To analyze FtsK assembly and translocation, and the subsequent activation of XerCD-dif recombination, we extended the tethered fluorophore motion technique, using two spectrally distinct fluorophores to monitor two effective lengths along the same tethered DNA molecule. We observed that FtsK assembled stepwise on DNA into a single hexamer, and began translocation rapidly (∼ 0.25 s). Without extruding DNA loops, single FtsK hexamers approached XerCD-dif and resided there for ∼ 0.5 s irrespective of whether XerCD-dif was synapsed or unsynapsed. FtsK then dissociated, rather than reversing. Infrequently, FtsK activated XerCD-dif recombination when it encountered a preformed synaptic complex, and dissociated before the completion of recombination, consistent with each FtsK-XerCD-dif encounter activating only one round of recombination.

Movement of the RecG Motor Domain upon DNA Binding Is Required for Efficient Fork Reversal.

RecG catalyzes reversal of stalled replication forks in response to replication stress in bacteria. The protein contains a fork recognition ("wedge") domain that binds branched DNA and a superfamily II (SF2) ATPase motor that drives translocation on double-stranded (ds)DNA. The mechanism by which the wedge and motor domains collaborate to catalyze fork reversal in RecG and analogous eukaryotic fork remodelers is unknown. Here, we used electron paramagnetic resonance (EPR) spectroscopy to probe conformational changes between the wedge and ATPase domains in response to fork DNA binding by Thermotoga maritima RecG. Upon binding DNA, the ATPase-C lobe moves away from both the wedge and ATPase-N domains. This conformational change is consistent with a model of RecG fully engaged with a DNA fork substrate constructed from a crystal structure of RecG bound to a DNA junction together with recent cryo-electron microscopy (EM) structures of chromatin remodelers in complex with dsDNA. We show by mutational analysis that a conserved loop within the translocation in RecG (TRG) motif that was unstructured in the RecG crystal structure is essential for fork reversal and DNA-dependent conformational changes. Together, this work helps provide a more coherent model of fork binding and remodeling by RecG and related eukaryotic enzymes.

Manipulating Electrical and Fluidic Access in Integrated Nanopore-Microfluidic Arrays Using Microvalves.

On-chip microvalves regulate electrical and fluidic access to an array of nanopores integrated within microfluidic networks. This configuration allows for on-chip sequestration of biomolecular samples in various flow channels and analysis by independent nanopores.

Self-Aligned Plasmonic Nanopores by Optically Controlled Dielectric Breakdown.

We present a novel cost-efficient method for the fabrication of high-quality self-aligned plasmonic nanopores by means of an optically controlled dielectric breakdown. Excitation of a plasmonic bowtie nanoantenna on a dielectric membrane localizes the high-voltage-driven breakdown of the membrane to the hotspot of the enhanced optical field, creating a nanopore that is automatically self-aligned to the plasmonic hotspot of the bowtie. We show that the approach provides precise control over the nanopore size and that these plasmonic nanopores can be used as single molecule DNA sensors with a performance matching that of TEM-drilled nanopores. The principle of optically controlled breakdown can also be used to fabricate nonplasmonic nanopores at a controlled position. Our novel fabrication process guarantees alignment of the nanopore with the optical hotspot of the nanoantenna, thus ensuring that pore-translocating biomolecules interact with the concentrated optical field that can be used for detection and manipulation of analytes.

Relevance of the Drag Force during Controlled Translocation of a DNA-Protein Complex through a Glass Nanocapillary.

Combination of glass nanocapillaries with optical tweezers allowed us to detect DNA-protein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA-protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries.