Unwinding Dynamics Research Articles

Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.

Dysregulated DnaB unwinding induces replisome decoupling and daughter strand gaps that are countered by RecA polymerization.

The replicative helicase, DnaB, is a central component of the replisome and unwinds duplex DNA coupled with immediate template-dependent DNA synthesis by the polymerase, Pol III. The rate of helicase unwinding is dynamically regulated through structural transitions in the DnaB hexamer between dilated and constricted states. Site-specific mutations in DnaB enforce a faster more constricted conformation that dysregulates unwinding dynamics, causing replisome decoupling that generates excess ssDNA and induces severe cellular stress. This surplus ssDNA can stimulate RecA recruitment to initiate recombinational repair, restart, or activation of the transcriptional SOS response. To better understand the consequences of dysregulated unwinding, we combined targeted genomic dnaB mutations with an inducible RecA filament inhibition strategy to examine the dependencies on RecA in mitigating replisome decoupling phenotypes. Without RecA filamentation, dnaB:mut strains had reduced growth rates, decreased mutagenesis, but a greater burden from endogenous damage. Interestingly, disruption of RecA filamentation in these dnaB:mut strains also reduced cellular filamentation but increased markers of double strand breaks and ssDNA gaps as detected by in situ fluorescence microscopy and FACS assays, TUNEL and PLUG, respectively. Overall, RecA plays a critical role in strain survival by protecting and processing ssDNA gaps caused by dysregulated helicase activity in vivo.

SsDNA reeling is an intermediate step in the reiterative DNA unwinding activity of the WRN-1 helicase

RecQ helicases are highly conserved between bacteria and humans. These helicases unwind various DNA structures in the 3' to 5'. Defective helicase activity elevates genomic instability and is associated with predisposition to cancer and/or premature aging. Recent single-molecule analyses have revealed the repetitive unwinding behavior of RecQ helicases from Escherichia coli to humans. However, the detailed mechanisms underlying this behavior are unclear. Here, we performed single-molecule studies of WRN-1 Caenorhabditis elegans RecQ helicase on various DNA constructs and characterized WRN-1 unwinding dynamics. We showed that WRN-1 persistently repeated cycles of DNA unwinding and rewinding with an unwinding limit of 25 to 31bp per cycle. Furthermore, by monitoring the ends of the displaced strand during DNA unwinding we demonstrated that WRN-1 reels in the ssDNA overhang in an ATP-dependent manner. While WRN-1 reeling activity was inhibited by a C.elegans homolog of human replication protein A, we found that C.elegans replication protein A actually switched the reiterative unwinding activity of WRN-1 to unidirectional unwinding. These results reveal that reeling-in ssDNA is an intermediate step in the reiterative unwinding process for WRN-1 (i.e., the process proceeds via unwinding-reeling-rewinding). We propose that the reiterative unwinding activity of WRN-1 may prevent extensive unwinding, allow time for partner proteins to assemble on the active region, and permit additional modulation invivo.

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.

Derivation of equations of motion of an unwinding cable considering transient-state tensile force in time-varying unwinding velocity

Unwinding dynamics refers to the study of nonlinear behaviour, tension profile, entangling, and fracturing in the process of unwinding a fibre from a package through the guide eyelet at a specific unwinding speed, to analyse the failure and nonlinear behaviours of unwinding. Although previous studies have dealt with transients, they have been limited to the transient process of a balloon with changes in boundary and tension only under a constant unwinding speed. In this study, the analysis method of an unwinding system at a time-varying unwinding speed was studied to analyse various unwinding conditions. The transient-state tension equation was derived by considering the effects of speed and acceleration at the time-varying unwinding speed. Then, the equation of motion derived in this study was verified through a comparison with the previous study.

Cooperative Analysis of Structural Dynamics in RNA-Protein Complexes by Single-Molecule Förster Resonance Energy Transfer Spectroscopy

RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.

Dynamic structural insights into the molecular mechanism of DNA unwinding by the bacteriophage T7 helicase

The hexametric T7 helicase (gp4) adopts a spiral lock-washer form and encircles a coil-like DNA (tracking) strand with two nucleotides bound to each subunit. However, the chemo-mechanical coupling mechanism in unwinding has yet to be elucidated. Here, we utilized nanotensioner-enhanced Förster resonance energy transfer with one nucleotide precision to investigate gp4-induced unwinding of DNA that contains an abasic lesion. We observed that the DNA unwinding activity of gp4 is hindered but not completely blocked by abasic lesions. Gp4 moves back and forth repeatedly when it encounters an abasic lesion, whereas it steps back only occasionally when it unwinds normal DNA. We further observed that gp4 translocates on the tracking strand in step sizes of one to four nucleotides. We propose that a hypothetical intermediate conformation of the gp4–DNA complex during DNA unwinding can help explain how gp4 molecules pass lesions, providing insights into the unwinding dynamics of gp4.

MutL sliding clamps coordinate exonuclease-independent Escherichia coli mismatch repair

A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites. Here we visualize the complete strand-specific excision process and find that long-lived EcMutL sliding clamps capture EcUvrD helicase near the ssDNA break, significantly increasing its unwinding processivity. EcSSB modulates the EcMutL–EcUvrD unwinding dynamics, which is rarely accompanied by extensive ssDNA exonuclease digestion. Together these observations are consistent with an exonuclease-independent MMR strand excision mechanism that relies on EcMutL–EcUvrD helicase-driven displacement of ssDNA segments between adjacent EcMutH–GATC incisions.

Asymmetric base-pair opening drives helicase unwinding dynamics

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.

Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase.

The RecQ family of helicases is highly conserved both structurally and functionally from bacteria to humans. Defects in human RecQ helicases are associated with genetic diseases that are characterized by cancer predisposition and/or premature aging. RecQ proteins exhibit 3′-5′ helicase activity and play critical roles in genome maintenance. Recent advances in single-molecule techniques have revealed the reiterative unwinding behavior of RecQ helicases. However, the molecular mechanisms involved in this process remain unclear, with contradicting reports. Here, we characterized the unwinding dynamics of the Caenorhabditis elegans RecQ helicase HIM-6 using single-molecule fluorescence resonance energy transfer measurements. We found that HIM-6 exhibits reiterative DNA unwinding and the length of DNA unwound by the helicase is sharply defined at 25–31 bp. Experiments using various DNA substrates revealed that HIM-6 utilizes the mode of ‘sliding back’ on the translocated strand, without strand-switching for rewinding. Furthermore, we found that Caenorhabditis elegans replication protein A, a single-stranded DNA binding protein, suppresses the reiterative behavior of HIM-6 and induces unidirectional, processive unwinding, possibly through a direct interaction between the proteins. Our findings shed new light on the mechanism of DNA unwinding by RecQ family helicases and their co-operation with RPA in processing DNA.

A model of DNA unwinding dynamics by the RecBCD complex and its regulation by Chi recognition

The Escherichia coli RecBCD enzyme is a heterotrimeric helicase-nuclease complex responsible for processing of double-stranded DNA breaks for repair by homologous recombination. It is a highly processive, duplex unwinding and degrading motor, with its activities being regulated by the octameric recombination hotspot, Chi, which is read as a single-stranded DNA sequence. Here, a model is presented for DNA unwinding by the RecBCD complex and its regulation by Chi recognition. With the model we study analytically the dynamics of DNA unwinding of both wild-type RecBCD and mutant RecBCDK177Q with the motor function of RecD being inactivated by mutagenesis, giving quantitative explanations of the available single-molecule experimental data. The peculiar features of RecBCD such as large variations of DNA unwinding speed of individual enzymes, sensitivity of unwinding speed of a RecBCD molecule on the change of environment, two translocase or helicase activities of RecBC and RecD, etc., are explained. Furthermore, predicted results are presented.

Dynamics of DNA unwinding by helicases with frequent backward steps

XPD (Xeroderma pigmentosum complementation group D) is a prototypical 5′ – 3′ translocating DNA helicase that exhibits frequent backward steps during DNA unwinding. Here, we propose a model of DNA unwinding by XPD. With the model we explain why XPD exhibits frequent backsteps while other helicases show rare backsteps. We explain quantitatively the single-molecule data on probability of –1-bp step and mean dwell time of one step versus ATP concentration for XPD at fixed large external force applied to the ends of the DNA hairpin to unzip the hairpin. We study DNA unwinding velocity, probability of –1-bp step and mean dwell time of one step for XPD versus external force at various ATP concentrations. We compare DNA unwinding dynamics of the 5′ – 3′ helicase XPD with that of 3′ – 5′ helicase RecQ. Our results show that the DNA unwinding velocity of XPD is sensitively dependent on the external force, which is contrast to RecQ that shows insensitive dependence of DNA unwinding velocity on the external force, explaining the experimental data showing that RecQ is an “optimally active” helicase while XPD is a “partially active” helicase. The DNA unwinding dynamics of different helicases under the external force is also studied.

Construction of unwinding equation of motion for thin cable in spherical coordinate system

The transient-state unwinding equation of motion for a thin cable can be derived by using Hamilton’s principle for an open system, which can consider the mass change produced by the unwinding velocity in a control volume. In general, most engineering problems can be analyzed in Cartesian, cylindrical, and spherical coordinate systems. In the field of unwinding dynamics, until now, only Cartesian and cylindrical coordinate systems have been used. A spherical coordinate system has not been used because of the complexity of derivatives. Therefore, in this study, the unwinding motion of a thin cable was analyzed using a spherical coordinate system in both water and air, and the results were compared with the results in Cartesian and cylindrical coordinate systems. The unwinding motions in the spherical, Cartesian, and cylindrical coordinate systems were nearly same in both water and air. The error related to the total length was within 0.5% in water, and the error related to the maximum balloon radius was also within 0.5 % in air. Therefore, it can be concluded that it is possible to solve the transient-state unwinding equation of motion in a spherical coordinate system.

Torque measurements during the spontaneous unbraiding of DNA molecules in the absence of pulling forces

We present an improved version of the magnetic tweezers/viscous drag (MTVD) hybrid assay that allows dynamic torque measurements during the spontaneous relaxation of DNA braids. The method relies on the ability of pairs of DNA molecules to rotate a microscopic probe attached to their upper ends using the elastic energy stored when braided. Digital tracking of the rotation of the dumbbell against the viscous drag allowed estimation of the torque exerted by the molecules. The unwinding dynamics of DNA braids against different amounts of viscous drag was characterized employing probes of two different sizes. We identified and characterized different perturbations that may affect the unbraiding dynamics. Estimates of the torques associated to the process of DNA unbraiding are reported.

Investigating the Enhancement of XPD Helicase Processivity by Single-Stranded Binding Protein RPA2

Ferroplasma acidarmanus helicase FacXPD is a Superfamily 2B helicase involved in transcription initiation and nucleotide excision repair. This archaeal protein serves as a model for understanding the molecular mechanisms of yeast Rad3 and human xeroderma pigmentosum group D protein (XPD). Previous work has shown that the unwinding of double-stranded DNA by FacXPD is enhanced by the single-stranded DNA binding protein FacRPA2. However, the mechanism by which unwinding enhancement occurs remains unknown. Here, we monitored the unwinding of a DNA hairpin by XPD in the presence of RPA2 using a single-molecule optical trap assay. By loading XPD and RPA2 onto DNA in a controlled sequence and by analyzing helicase unwinding dynamics, we distinguish between different potential mechanisms of regulation. Our data point toward a model of processivity enhancement in which RPA2 transiently interacts with the XPD-DNA complex and stabilizes a faster, more processive state of the helicase.

Dynamics of monomeric and hexameric helicases

Helicases are a ubiquitous class of enzymes that use the energy of ATP hydrolysis to unwind nucleic acid (NA) duplex. According to the structures, helicases can be classified as the non-ring-shaped (or monomeric) and ring-shaped (or hexameric). To understand the NA unwinding mechanism, here we study theoretically the unwinding dynamics of both the monomeric and hexameric helicases based on our proposed model. Various available single-molecule experimental data on unwinding speed of both the monomeric and hexameric helicases versus the external force applied to the ends of the NA duplex to unzip the duplex or versus the stability of the NA duplex are consistently and quantitatively explained. We provide quantitative explanations of the experimental data showing that while the unwinding speeds of some monomeric helicases are insensitively dependent on the external force they are sensitively dependent on the stability of the NA duplex. The experimental data showing that wild-type Rep translocates along ssDNA with a lower speed than RepΔ2B (removal of the 2B subdomain of Rep) and that RepΔ2B monomer can unwind DNA whereas the wild-type monomer is unable to unwind DNA are also quantitatively explained. Our studies indicate that although the monomeric and hexameric helicases show very different features on the dependence of NA unwinding speed upon the external force, they use much similar active mechanisms to unwind NA duplex.

RNA Unwinding by the Helicase Mtr4p and the TRAMP Complex Investigated via High-Resolution Optical Trapping

We present single-molecule high-resolution optical trapping measurements of RNA unwinding by the Ski2-like nuclear helicase Mtr4p both alone and within the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex. RNA helicases such as Mtr4p play key roles in RNA metabolism. Mtr4p is thought to assist RNA degradation by the exosome presumably by unwinding RNA structures. In addition, the TRAMP complex appends short 3′ poly(A) tails to RNA meant for degradation by the exosome. Mtr4p within the TRAMP complex plays a crucial role in regulating the polyadenylation activity, limiting the poly(A) tail length. The unwinding activity of Mtr4p is required and indeed within the TRAMP complex, Mtr4p unwinds duplex RNA 10x faster than when alone. However, the dynamic details of the RNA unwinding activity and interaction of Mtr4p and TRAMP and indeed the Ski2-like family of RNA helicases are unknown. We have performed high-resolution RNA hairpin unwinding experiments for both Mtr4p and TRAMP. We find that Mtr4p unwinds RNA duplexes in a single or multiple bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4p remains stably bound after unwinding the duplex, preventing re-annealing. TRAMP unwinding experiments reveal a faster molecular recognition by the Mtr4p helicase when in complex as well as enhanced RNA duplex unwinding dynamics. Combined high-resolution trapping and fluorescence experiments underway may reveal how the competition within TRAMP between the 3′-to-5′ Mtr4p helicase and the 5′-to-3′ Trf4p polymerase is resolved dynamically.

Unwinding Dynamics of a Helically Wrapped Polymer

We study the rotational dynamics of a flexible polymer initially wrapped around a rigid rod and unwinding from it. This dynamics is of interest in several problems in biology and constitutes a fundamental instance of polymer relaxation from a state of minimal entropy. We investigate the dynamics of several quantities such as the total and local winding angles and metric quantities. The results of simulations performed in two and three dimensions, with and without self-avoidance, are explained by a theory based on scaling arguments and on a balance between frictional and entropic forces. The early stage of the dynamics is particularly rich, being characterized by three coexisting phases.

Supercoiled pseudocircular domains in single‐twisted DNAs under tension: Elastic constants and unwinding dynamics in complexes with topo I

Extension versus twist data of Koster et al. (Nature 2005, 434, 671-674) are analyzed to obtain C for the main-chain segments and the twist energy parameter (ET ) for the supercoiled pseudocircular (sp) domain(s) from which C is estimated via simulations. The torsional rigidity in the tension-free sp domain(s) (C = 163 fJ fm) is typical of the unstrained DNA and is less than half the value in the main-chain segments under tension (C = 350-410 fJ fm). Tension is suggested to induce a structural transition to a torsionally stiffer state. Data of Koster et al. for the rate of extension owing to unwinding of a covalent complex of DNA with human Topoisomerase Ib (H Topo I) are analyzed to determine the torque and rate of rotation from which an effective friction coefficient is obtained. A Langevin equation for the unwinding motion in a supercoiled DNA:H Topo I complex is solved to obtain the temporal trajectory of the average winding angle and the time-dependent distribution of winding angles. The mean rate constant for the religation reaction is estimated from the measured probability of reaction per turn. We predict that unwinding proceeds rather far during a single-cleavage and religation cycle, and is effectively completely equilibrated during the 3.2 cleavage and religation cycles that occur during each noncovalent binding and dissociation event. H Topo I is predicted to be completely processive as in accord with observations on calf-thymus Topo I (Brewood et al., Biochemistry 2010, 49, 3367-3380).

Single-Molecule Imaging of the Oligomer Formation of the Nonhexameric Escherichia coli UvrD Helicase

Superfamily I helicases are nonhexameric helicases responsible for the unwinding of nucleic acids. However, whether they unwind DNA in the form of monomers or oligomers remains a controversy. In this study, we addressed this question using direct single-molecule fluorescence visualization of Escherichia coli UvrD, a superfamily I DNA helicase. We performed a photobleaching-step analysis of dye-labeled helicases and determined that the helicase is bound to 18-basepair (bp) double-stranded DNA (dsDNA) with a 3′ single-stranded DNA (ssDNA) tail (12, 20, or 40 nt) in a dimeric or trimeric form in the absence of ATP. We also discovered through simultaneous visualization of association/dissociation of the helicase with/from DNA and the DNA unwinding dynamics of the helicase in the presence of ATP that these dimeric and trimeric forms are responsible for the unwinding of DNA. We can therefore propose a new kinetic scheme for the helicase-DNA interaction in which not only a dimeric helicase but also a trimeric helicase can unwind DNA. This is, to our knowledge, the first direct single-molecule nonhexameric helicase quantification study, and it strongly supports a model in which an oligomer is the active form of the helicase, which carries important implications for the DNA unwinding mechanism of all superfamily I helicases.