Unwinding Process Research Articles

A single-molecule approach to unravel the molecular mechanism of the action of Deinococcus radiodurans RecD2 and its interaction with SSB and RecA in DNA repair

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. They are responsible for resolving double-stranded DNA (dsDNA) into single-strands, which is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions to the organism's unusually high tolerance to gamma radiation and hydrogen peroxide. Although the results from X-ray Crystallography studies have revealed the structural characteristics of the protein, direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5′ terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

Fading-free Φ-OTDR evaluation based on the statistical analysis of phase hopping.

In phase-sensitive optical time domain reflectometry (Φ-OTDR), false phase peaks caused by interference fading have been observed experimentally; however, the statistical law has not yet been disclosed. In this work, after clarifying that the false phase peaks originate from the phase hopping of demodulated phase noise during the unwinding process, we define the phase hopping rate (PHR) to evaluate the degree of fading and study the quantitative relationship between the PHR and signal-to-noise ratio (SNR) of the measured signal through theoretical derivation and experimental verification. In addition, a moving rotated-vector-average (MRVA) method is proposed to suppress the fading and eliminate the false phase peaks. In the experiment, after MRVA processing with a 25 ns sliding window, the lowest SNR is pulled from 0.003 to 14.9, and the corresponding PHR is reduced from 0.237 to less than 0.0001, which is consistent with the theoretical analysis.

Probing the structure and function of polymerase θ helicase-like domain

DNA Polymerase θ is the key actuator of the recently identified double-strand break repair pathway, theta-mediated end joining (TMEJ). It is the only known polymerase to have a 3-domain architecture containing an independently functional family A DNA polymerase tethered by a long central region to an N-terminal helicase-like domain (HLD). Full-length polymerase θ and the isolated HLD hydrolyze ATP in the presence of DNA, but no processive DNA duplex unwinding has been observed. Based on sequence and structure conservation, the HLD is classified as a member of helicase superfamily II and, more specifically, the Ski2-like family. The specific subdomain composition and organization most closely resemble that of archaeal DNA repair helicases Hel308 and Hjm. The underlying structural basis as to why the HLD is not able to processively unwind duplex DNA, despite its similarity to bona fide helicases, remains elusive. Activities of the HLD include ATP hydrolysis, protein displacement, and annealing of complementary DNA. These observations have led to speculation about the role of the HLD within the context of double-strand break repair via TMEJ, such as removal of single-stranded DNA binding proteins like RPA and RAD51 and microhomology alignment. This review summarizes the structural classification and organization of the polymerase θ HLD and its homologs and explores emerging data on its biochemical activities. We conclude with a simple, speculative model for the HLD’s role in TMEJ.

Hierarchical self-assembling and helical structure in focal conic domains in meniscus of ferroelectric liquid crystal.

We investigate experimentally the formation of focal conic domains of the ferroelectric phase of a liquid crystal, chiral smectic C (SmC^{*}), in the meniscus geometry. The meniscus geometry is formed in the gap between two glass plates which are placed on a common substrate. This gap is called here a physical cavity. Focal conic domains (FCDs) in the physical cavity with dimensions of micrometer scale are investigated under an optical polarizing microscope which enables us to extract the information on the helical structure formation in the constraint and gradient topological meniscus interface. The helical pitch in the FCD is observed to be shorter than in planar confined geometry. A crucial phenomenon of unwrapping and wrapping of helical structure from one FCD to another is also observed. In-plane application of an electric field on a FCD revealed the asymmetric helical unwinding process whereas an increase in temperature has shown symmetrical unwinding. The helical structure based observation is significant for understanding the ferroelectric phase in focal conic domains and their application in microlenses and optical components.

Single-Molecule Insights Into the Dynamics of Replicative Helicases.

Helicases are molecular motors that translocate along single-stranded DNA and unwind duplex DNA. They rely on the consumption of chemical energy from nucleotide hydrolysis to drive their translocation. Specialized helicases play a critically important role in DNA replication by unwinding DNA at the front of the replication fork. The replicative helicases of the model systems bacteriophages T4 and T7, Escherichia coli and Saccharomyces cerevisiae have been extensively studied and characterized using biochemical methods. While powerful, their averaging over ensembles of molecules and reactions makes it challenging to uncover information related to intermediate states in the unwinding process and the dynamic helicase interactions within the replisome. Here, we describe single-molecule methods that have been developed in the last few decades and discuss the new details that these methods have revealed about replicative helicases. Applying methods such as FRET and optical and magnetic tweezers to individual helicases have made it possible to access the mechanistic aspects of unwinding. It is from these methods that we understand that the replicative helicases studied so far actively translocate and then passively unwind DNA, and that these hexameric enzymes must efficiently coordinate the stepping action of their subunits to achieve unwinding, where the size of each step is prone to variation. Single-molecule fluorescence microscopy methods have made it possible to visualize replicative helicases acting at replication forks and quantify their dynamics using multi-color colocalization, FRAP and FLIP. These fluorescence methods have made it possible to visualize helicases in replication initiation and dissect this intricate protein-assembly process. In a similar manner, single-molecule visualization of fluorescent replicative helicases acting in replication identified that, in contrast to the replicative polymerases, the helicase does not exchange. Instead, the replicative helicase acts as the stable component that serves to anchor the other replication factors to the replisome.

Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity.

DNA replication complexes (replisomes) frequently encounter barriers that can eject them prematurely from the genome. To avoid the lethality of incomplete DNA replication that arises from these events, bacteria have evolved “DNA replication restart” mechanisms to reload replisomes onto abandoned replication forks. The Escherichia coli PriA DNA helicase orchestrates this process by recognizing and remodeling replication forks and recruiting additional proteins that help to drive replisome reloading. We have identified a conserved sequence motif within a linker region of PriA that docks into a groove on the exterior of the PriA helicase domain. Alterations to the motif reduce the apparent processivity and attenuate structure-specific helicase activity in PriA, implicating the motif as a potential autoregulatory element in replication fork processing. The study also suggests that multiple PriA molecules may function in tandem to enhance DNA unwinding processivity, highlighting an unexpected similarity between PriA and other DNA helicases.

Modelling of tension in yarn package unwinding

Yarn unwinding from a package is an essential step in many textile processes. The quality of the yarn is numericallyexpressed mainly by values of mechanical quantities. In the unwinding process viscoelastic properties are the mostimportant ones. They depend on how the yarn is stressed. The quality of the yarn that is being unwound should not bereduced, unless this reduction doesn’t significantly lower the quality of the textile fabric. During unwind the yarn tensionis not constant, but it oscillates within some interval. Even when the yarn is not strongly stressed, the yarn still can breaksometimes. This is why we think that a cross-wound package is not an ideal form of a package and that such packagesaren’t always made without flaws. We strive to achieve as large warping and weaving speeds as possible, therefore ouraim is to improve the theory of cross-wound package unwinding and to find the necessary modifications of the yarnunwinding process.

Single-molecule mechanics of synthetic aromatic amide helices: Ultrafast and robust non-dissipative winding

Summary Because of proteins’ many degrees of conformational freedom, programming protein folding dynamics, overall elasticity, and motor functions remains an elusive objective. Instead, smaller and simpler objects, such as synthetic foldamers, may be amenable to design. However, little is known about their mechanical performance. Here, we show that reducing molecular size may not compromise mechanical properties. We report that helical aromatic oligoamides as small as 1 nm possess outstanding elasticity and outperform most natural helices. Using single-molecule force spectroscopy, we characterize their folding trajectories and intermediate states. We show that they cooperatively and reversibly unwind at high forces. They extend up to 3.8 times their original length and rewind against considerable forces on a timescale of 10 μs. Pulling and relaxing cycles follow the same trace up to a very high loading rate, indicating that the mechanical energy accumulated during the stretching does not dissipate and is immediately reusable.

Verification of numerical analysis for unwinding cable with time-varying unwinding velocity

In this study, the nonlinear unwinding behavior of cables in a transient state wound in a cylindrical spool package was analyzed using unwinding dynamics. In previous studies, investigations were conducted using constant unwinding velocity. However, this method is limited due to complexities regarding the analysis of the actual behavior where the unwinding velocity increases or decreases. In this paper, to prevent problems, such as twisting, entanglement, and cutting, that occur during the rapid unwinding process, an experimental device that converts unwinding velocity over time was developed. Using a cable unwinding system, high-speed camera, and tension sensor, the equation of motion, which was derived in a previous study using the expanded Hamilton theorem, was experimentally verified. Moreover, the cable’s tension change and behavior in the unwinding process were analyzed, and a method to solve the unwinding problem was studied.

Residue-Level Functional Couplings and their Effect on RNA Remodelling by the Zika Virus NS3 Helicase

Nucleic acid unwinding carried out by helicases is achieved by efficient coupling of the ATPase hydrolysis to binding, translocation and local melting of the duplexed strands. Understanding this functional coupling between the active sites of the helicase can yield insight into mechanisms that determine the efficiency of RNA remodeling by the helicases from Flaviviridae NS3 family. We employed weighted energy contact networks (wECN) to compare the topology of the NS3 helicases (NS3h) from Zika virus with Hepatitis C virus and Dengue virus. This complemented with fluorescence based binding and unwinding assays allowed us to characterize the dynamics of RNA remodeling events by the ZIKV NS3h and distinguish it from the NS3 helicase family. ZIKV NS3h uniquely unwinds partial duplex RNA 3’ overhangs albeit with low processivity, and its ATPase activity is not stimulated by RNA binding. Using the wECN network model for ZIKA NS3h, we identified residues distal to the binding sites that strongly exert an influence on the RNA unwinding activity and processivity. Analysis of designed mutants with enhanced RNA unwinding activity suggests a contrasting mechanism that dictates helicase efficiency in ZIKV virus NS3h as compared to HCV and DENV, where enhancement is achieved by improving ATPase efficiency. Our assays also allowed us to characterize RNA annealing, a second mode of RNA remodeling mediated by the ZIKV NS3 helicase. The effect of mutations on RNA annealing further provided insights into factors that allow the NS3 protein to sequester RNA at low concentrations. Our mechanistic insights into RNA remodeling events by NS3h will allow rational design of tuned RNA helicases and identify inhibitors for replication by the Zika virus.

Force-Dependent Stimulation of RNA Unwinding by SARS-CoV-2 NSP13 Helicase

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, the mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism.

The KH domain facilitates the substrate specificity and unwinding processivity of DDX43 helicase

The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation–seq, and cross-linking immunoprecipitation–seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

The superfamily 1 helicase nonstructural protein 13 (nsp13) is required for SARS-CoV-2 replication. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the RNA substrate. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be activated >50-fold by piconewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, such as hepatitis C virus NS3, and instead draws stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

Assignment of the Ile, Leu, Val, Met and Ala methyl group resonances of the DEAD-box RNA helicase DbpA from E. coli

ATP-dependent DEAD-box helicases constitute one of the largest families of RNA helicases and are important regulators of most RNA-dependent cellular processes. The functional core of these enzymes consists of two RecA-like domains. Changes in the interdomain orientation of these domains upon ATP and RNA binding result in the unwinding of double-stranded RNA. The DEAD-box helicase DbpA from E. coli is involved in ribosome maturation. It possesses a C-terminal RNA recognition motif (RRM) in addition to the canonical RecA-like domains. The RRM recruits DbpA to nascent ribosomes by binding to hairpin 92 of the 23S rRNA. To follow the conformational changes of Dbpa during the catalytic cycle we initiated solution state NMR studies. We use a divide and conquer approach to obtain an almost complete resonance assignment of the isoleucine, leucine, valine, methionine and alanine methyl group signals of full length DbpA (49 kDa). In addition, we also report the backbone resonance assignments of two fragments of DbpA that were used in the course of the methyl group assignment. These assignments are the first step towards a better understanding of the molecular mechanism behind the ATP-dependent RNA unwinding process catalyzed by DEAD-box helicases.

Monitoring Enzymatic RNA G-Quadruplex Unwinding Activities by Nuclease Sensitivity and Reverse Transcription Stop Assays.

Multiple different methods have been employed to investigate the unwinding of RNA G-quadruplexes by various helicase proteins. Each has their own pitfalls, namely, looking at non-native or chemically modified RNA sequences, biasing the unwinding process with competing trap nucleotides, and a lack of context sequence to the 5' and 3' of the RNA G-quadruplex structure. Herein we present two straightforward methods that allow for quadruplex unwinding to be monitored on native RNA sequences without the use of fluorescent modifications, specialized equipment, or trap nucleotides to be employed.

Tertiary Base Triple Formation in the SRV-1 Frameshifting Pseudoknot Stabilizes Secondary Structure Components.

Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot are essential in stimulating -1 ribosomal frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus ribosomal frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA-RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1-loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1-loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.

Nanomechanical Properties of a Supramolecular Helix Stabilized by Non-Covalent Interactions.

Supramolecular helices have unique properties and many potential applications, such as chiral separation and asymmetric catalysis. Mechanical property (stability) of the supramolecular helix plays important roles in their functions. Due to the limitation of detection method, it is quite challenging to investigate nanomechanical properties of individual supramolecular helices stabilized by pure supramolecular interactions. Here atomic force microscopy (AFM)-based single molecule force spectroscopy (SMFS) is used to study the nanomechanical properties of a thermal-responsive supramolecular helix. The unwinding force plateau is observed in the force-extension curve, and the rupture force of the helix is dependent on the loading rate. In addition, the force-induced unwinding process is reversible and there is almost no energy dissipation in the process. Furthermore, the result of thermal shape-fluctuation analysis shows that the persistence length of the supramolecular helix is about 222nm, which is much larger than helical structure formed by double-stranded DNA (dsDNA). However, because of its unique backbone structure, the supramolecular helix exhibits higher dynamic flexibility during force-induced deformation, since the persistence length determined from the stretching experiment is much smaller (1.1nm).

A variant of the unknotting number of a knot with applications to the topology of biomolecules

The creation of unwound or unknotted loops of DNA is facilitated by topoisomerases, which alter the conformation of DNA by cleaving the phosphate backbone on one or both the strands. A major unexplored question has been the possibility of topoisomerases failing to unpack supercoiled DNA on any one of the several knots, thereby failing to initiate the unwinding process. Computing the unknotting number enables biologists to estimate the smallest amount of times the topoisomerase must act to unpack DNA. In this paper, we define a new variant called the fault-tolerant unknotting number t(K) of the unknotting number u(K) for a knot K. Besides, t(K) is related to the unknotting and knotting phenomena of DNA. We determine t(D) for some families of knot diagrams and conclude that the difference between u(D) and t(D) for a knot diagram D can be arbitrarily large. We also conjectured the existence of fault-tolerant unknotting number for every knot diagram and every knot.

Terahertz Wave Accelerates DNA Unwinding: A Molecular Dynamics Simulation Study.

Unwinding the double helix of the DNA molecule is the basis of gene duplication and gene editing, and the acceleration of this unwinding process is crucial to the rapid detection of genetic information. Based on the unwinding of six-base-pair DNA duplexes, we demonstrate that a terahertz stimulus at a characteristic frequency (44.0 THz) can serve as an efficient, nonthermal, and long-range method to accelerate the unwinding process of DNA duplexes. The average speed of the unwinding process increased by 20 times at least, and its temperature was significantly reduced. The mechanism was revealed to be the resonance between the terahertz stimulus and the vibration of purine connected by the weak hydrogen bond and the consequent break in hydrogen bond connections between these base pairs. Our findings potentially provide a promising application of terahertz technology for the rapid detection of nucleic acids, biomedicine, and therapy.

Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor.

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase–nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5′ to 3′ ssDNA translocase and 5′ to 3′ helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase–nucleases and DNA looping motor proteins in general.