scholarly journals Submicrometer elasticity of double-stranded DNA revealed by precision force-extension measurements with magnetic tweezers

2019 ◽  
Vol 5 (6) ◽  
pp. eaav1697 ◽  
Author(s):  
Min Ju Shon ◽  
Sang-Hyun Rah ◽  
Tae-Young Yoon
Keyword(s):  
Single Molecule ◽  
Persistence Length ◽  
Gc Content ◽  
Magnetic Tweezers ◽  
Force Variability ◽  
Chain Model ◽  
Force Extension ◽  
Dna Hairpins ◽  
Double Stranded Dna ◽  
Order Of Magnitude

Submicrometer elasticity of double-stranded DNA (dsDNA) governs nanoscale bending of DNA segments and their interactions with proteins. Single-molecule force spectroscopy, including magnetic tweezers (MTs), is an important tool for studying DNA mechanics. However, its application to short DNAs under 1 μm is limited. We developed an MT-based method for precise force-extension measurements in the 100-nm regime that enables in situ correction of the error in DNA extension measurement, and normalizes the force variability across beads by exploiting DNA hairpins. The method reduces the lower limit of tractable dsDNA length down to 198 base pairs (bp) (67 nm), an order-of-magnitude improvement compared to conventional tweezing experiments. Applying this method and the finite worm-like chain model we observed an essentially constant persistence length across the chain lengths studied (198 bp to 10 kbp), which steeply depended on GC content and methylation. This finding suggests a potential sequence-dependent mechanism for short-DNA elasticity.

scholarly journals Sequence-dependent Three Interaction Site (TIS) Model for Single and Double-stranded DNA

2018 ◽  
Author(s):  
Debayan Chakraborty ◽  
Naoto Hori ◽  
D. Thirumalai
Keyword(s):  
Electrostatic Interactions ◽  
Persistence Length ◽  
Data Bank ◽  
Coarse Grained ◽  
Force Extension ◽  
Sequence Dependence ◽  
Coarse Grained Model ◽  
Interaction Sites ◽  
Double Stranded Dna ◽  
Centers Of Mass

AbstractWe develop a robust coarse-grained model for single and double stranded DNA by representing each nucleotide by three interaction sites (TIS) located at the centers of mass of sugar, phosphate, and base. The resulting TIS model includes base-stacking, hydrogen bond, and electrostatic interactions as well as bond-stretching and bond angle potentials that account for the polymeric nature of DNA. The choices of force constants for stretching and the bending potentials were guided by a Boltzmann inversion procedure using a large representative set of DNA structures extracted from the Protein Data Bank. Some of the parameters in the stacking interactions were calculated using a learning procedure, which ensured that the experimentally measured melting temperatures of dimers are faithfully reproduced. Without any further adjustments, the calculations based on the TIS model reproduces the experimentally measured salt and sequence dependence of the size of single stranded DNA (ssDNA), as well as the persistence lengths of poly(dA) and poly(dT) chains. Interestingly, upon application of mechanical force the extension of poly(dA) exhibits a plateau, which we trace to the formation of stacked helical domains. In contrast, the force-extension curve (FEC) of poly(dT) is entropic in origin, and could be described by a standard polymer model. We also show that the persistence length of double stranded DNA, formed from two complementary ssDNAs with one hundred and thirty base pairs, is consistent with the prediction based on the worm-like chain. The persistence length, which decreases with increasing salt concentration, is in accord with the Odijk-Skolnick-Fixman theory intended for stiff polyelectrolyte chains near the rod limit. The range of applications, which did not require adjusting any parameter after the initial construction based solely on PDB structures and melting profiles of dimers, attests to the transferability and robustness of the TIS model for ssDNA and dsDNA.

scholarly journals Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

2019 ◽  
Author(s):  
Hazal B. Kose ◽  
Sherry Xie ◽  
George Cameron ◽  
Melania S. Strycharska ◽  
Hasan Yardimci
Keyword(s):  
Single Molecule ◽  
Replication Fork ◽  
Double Helix ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Replicative Helicase ◽  
Double Stranded Dna ◽  
Order Of Magnitude ◽  
Dna Strand ◽  
Dna Double Helix

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

scholarly journals Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

2019 ◽  
Author(s):  
Alberto Marin-Gonzalez ◽  
Cesar L. Pastrana ◽  
Rebeca Bocanegra ◽  
Alejandro Martín-González ◽  
J.G. Vilhena ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mechanical Response ◽  
Magnetic Tweezers ◽  
Crystallographic Structure ◽  
Chain Model ◽  
Microscopy Imaging ◽  
Length Scales

ABSTRACTA-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range (∼200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

Characterizing titin's I-band Ig domain region as an entropic spring

1998 ◽  
Vol 111 (11) ◽  
pp. 1567-1574 ◽  
Author(s):  
W.A. Linke ◽  
M.R. Stockmeier ◽  
M. Ivemeyer ◽  
H. Hosser ◽  
P. Mundel
Keyword(s):  
Muscle Protein ◽  
Persistence Length ◽  
Thick Filament ◽  
Contour Length ◽  
Chain Model ◽  
Bending Rigidity ◽  
Force Extension ◽  
Average Force ◽  
Wormlike Chain ◽  
C Terminus

The poly-immunoglobulin domain region of titin, located within the elastic section of this giant muscle protein, determines the extensibility of relaxed myofibrils mainly at shorter physiological lengths. To elucidate this region's contribution to titin elasticity, we measured the elastic properties of the N-terminal I-band Ig region by using immunofluorescence/immunoelectron microscopy and myofibril mechanics and tried to simulate the results with a model of entropic polymer elasticity. Rat psoas myofibrils were stained with titin-specific antibodies flanking the Ig region at the N terminus and C terminus, respectively, to record the extension behaviour of that titin segment. The segment's end-to-end length increased mainly at small stretch, reaching approximately 90% of the native contour length of the Ig region at a sarcomere length of 2.8 microm. At this extension, the average force per single titin molecule, deduced from the steady-state passive length-tension relation of myofibrils, was approximately 5 or 2.5 pN, depending on whether we assumed a number of 3 or 6 titins per half thick filament. When the force-extension curve constructed for the Ig region was simulated by the wormlike chain model, best fits were obtained for a persistence length, a measure of the chain's bending rigidity, of 21 or 42 nm (for 3 or 6 titins/half thick filament), which correctly reproduced the curve for sarcomere lengths up to 3.4 microm. Systematic deviations between data and fits above that length indicated that forces of >30 pN per titin strand may induce unfolding of Ig modules. We conclude that stretches of at least 5–6 Ig domains, perhaps coinciding with known super repeat patterns of these titin modules in the I-band, may represent the unitary lengths of the wormlike chain. The poly-Ig regions might thus act as compliant entropic springs that determine the minute levels of passive tension at low extensions of a muscle fiber.

scholarly journals Denaturation transition of stretched DNA

2013 ◽  
Vol 41 (2) ◽  
pp. 639-645 ◽  
Author(s):  
Andreas Hanke
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Present Review ◽  
Dna Molecules ◽  
Force Measurements ◽  
Force Extension ◽  
Atomic Force Spectroscopy ◽  
Atomic Force

In the last two decades, single-molecule force measurements using optical and magnetic tweezers and atomic force spectroscopy have dramatically expanded our knowledge of nucleic acids and proteins. These techniques characterize the force on a biomolecule required to produce a given molecular extension. When stretching long DNA molecules, the observed force–extension relationship exhibits a characteristic plateau at approximately 65 pN where the DNA may be extended to almost twice its B-DNA length with almost no increase in force. In the present review, I describe this transition in terms of the Poland–Scheraga model and summarize recent related studies.

scholarly journals Single-Molecule Micromanipulation Studies of Methylated DNA

2020 ◽  
Author(s):  
T. Zaichuk ◽  
J. F. Marko
Keyword(s):  
Structural Stability ◽  
Single Molecule ◽  
Cytosine Methylation ◽  
Mechanical Stability ◽  
Persistence Length ◽  
Epigenetic Mark ◽  
Force Extension ◽  
Dna Flexibility ◽  
Methylated Dna ◽  
High Structural Stability

AbstractCytosine methylated at the 5-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded “bubbles” of DNA, under physiologically relevant stretching forces and supercoiling densities. The stiffness and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.Statement of SignificanceDespite countless structural and functional studies of DNA methylation, a key epigenetic mark in higher organisms, research towards the understanding of DNA intrinsic structural properties in the context of methylation layout representing different epigenetic landscapes is still in its initial stage. We utilize single molecule spectroscopy to analyze the effect of sparse symmetric and asymmetric 5-mC modification on the mechanical stability of long double-stranded DNA. Our findings establish that at physiologically relevant forces and supercoiling densities increased DNA flexibility of non-CpG methylated DNA translates to the high structural stability.

scholarly journals Extensions of the worm-like-chain model to tethered active filaments under tension

2020 ◽  
Author(s):  
Xinyu Liao ◽  
Prashant K. Purohit ◽  
Arvind Gopinath
Keyword(s):  
Boundary Conditions ◽  
Single Molecule ◽  
Molecular Motors ◽  
Optical Traps ◽  
Chain Model ◽  
Mean Square ◽  
Force Extension ◽  
Space Correlation ◽  
Various Boundary Conditions ◽  
Random Forces

Intracellular elastic filaments such as microtubules are subject to thermal Brownian noise and active noise generated by molecular motors that convert chemical energy into mechanical work. Similarly, polymers in living fluids such as bacterial suspensions and swarms suffer bending deformations as they interact with single bacteria or with cell clusters. Often these filaments perform mechanical functions and interact with their networked environment through cross-links, or have other similar constraints placed on them. Here we examine the mechanical properties - under tension - of such constrained active filaments under canonical boundary conditions motivated by experiments. Fluctuations in the filament shape are a consequence of two types of random forces - thermal Brownian forces, and activity derived forces with specified time and space correlation functions. We derive force-extension relationships and expressions for the mean square deflections for tethered filaments under various boundary conditions including hinged and clamped constraints. The expressions for hinged-hinged boundary conditions are reminiscent of the worm-like-chain model and feature effective bending moduli and mode-dependent non-thermodynamic effective temperatures controlled by the imposed force and by the activity. Our results provide methods to estimate the activity by measurements of the force-extension relation of the filaments or their mean-square deflections which can be routinely performed using optical traps, tethered particle experiments, or other single molecule techniques.

scholarly journals Sugar-Pucker Force-Induced Transition in Single-Stranded DNA

2021 ◽  
Vol 22 (9) ◽  
pp. 4745
Author(s):  
Xavier Viader-Godoy ◽  
Maria Manosas ◽  
Felix Ritort
Keyword(s):  
Optical Tweezers ◽  
Conformational Transition ◽  
Persistence Length ◽  
Elastic Response ◽  
Contour Length ◽  
Dna Unwinding ◽  
Chain Model ◽  
Force Extension ◽  
Single Stranded Dna ◽  
Sugar Pucker

The accurate knowledge of the elastic properties of single-stranded DNA (ssDNA) is key to characterize the thermodynamics of molecular reactions that are studied by force spectroscopy methods where DNA is mechanically unfolded. Examples range from DNA hybridization, DNA ligand binding, DNA unwinding by helicases, etc. To date, ssDNA elasticity has been studied with different methods in molecules of varying sequence and contour length. A dispersion of results has been reported and the value of the persistence length has been found to be larger for shorter ssDNA molecules. We carried out pulling experiments with optical tweezers to characterize the elastic response of ssDNA over three orders of magnitude in length (60–14 k bases). By fitting the force-extension curves (FECs) to the Worm-Like Chain model we confirmed the above trend:the persistence length nearly doubles for the shortest molecule (60 b) with respect to the longest one (14 kb). We demonstrate that the observed trend is due to the different force regimes fitted for long and short molecules, which translates into two distinct elastic regimes at low and high forces. We interpret this behavior in terms of a force-induced sugar pucker conformational transition (C3′-endo to C2′-endo) upon pulling ssDNA.

scholarly journals Ring-shaped replicative helicase encircles double-stranded DNA during unwinding

2019 ◽  
Vol 47 (21) ◽  
pp. 11344-11354 ◽  
Author(s):  
Sihwa Joo ◽  
Bong H Chung ◽  
Mina Lee ◽  
Tai H Ha
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Model Systems ◽  
Bovine Papillomavirus ◽  
Viral Origin ◽  
Replicative Helicase ◽  
Single Molecule Level ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Cellular Dna

Abstract Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26–30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

scholarly journals Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

2020 ◽  
Vol 48 (9) ◽  
pp. 5024-5036
Author(s):  
Alberto Marin-Gonzalez ◽  
Cesar L Pastrana ◽  
Rebeca Bocanegra ◽  
Alejandro Martín-González ◽  
J G Vilhena ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Sequences ◽  
Mechanical Response ◽  
Magnetic Tweezers ◽  
Crystallographic Structure ◽  
Chain Model ◽  
Microscopy Imaging ◽  
Wide Range

Abstract A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

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