scholarly journals DNA fluctuations reveal the size and dynamics of topological domains

2021 ◽  
Author(s):  
Willem Vanderlinden ◽  
Enrico Skoruppa ◽  
Pauline J. Kolbeck ◽  
Enrico Carlon ◽  
Jan Lipfert
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Torsional Stiffness ◽  
Molecular Architecture ◽  
Supercoiled Dna ◽  
Topological Domains ◽  
Single Molecule Level ◽  
Processing Enzymes

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein induced topological domains in DNA, quantitative and time-resolved approaches are required. Here we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real-time and at the single molecule level. Our approach is based on quantifying the extension fluctuations -in addition to the mean extension- of supercoiled DNA in magnetic tweezers. Using a combination of high-speed magnetic tweezers experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how transient (partial) dissociation of DNA bridging proteins results in dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our approach to enable quantification of the dynamics and reaction pathways of DNA processing enzymes and motor proteins, in the context of physiologically relevant forces and supercoiling densities.

scholarly journals Supercoiling DNA optically

2019 ◽  
Vol 116 (52) ◽  
pp. 26534-26539
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Supercoiling ◽  
Biological Interactions ◽  
Mitochondrial Transcription ◽  
Supercoiled Dna ◽  
Single Molecule Level ◽  
Mitochondrial Transcription Factor ◽  
Wide Range

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

scholarly journals Condensin-driven loop extrusion on supercoiled DNA

2021 ◽  
Author(s):  
Eugene Kim ◽  
Alejandro Martin Gonzalez ◽  
Biswajit Pradhan ◽  
Jaco van der Torre ◽  
Cees Dekker
Keyword(s):  
Single Molecule ◽  
Molecular Motor ◽  
Time Lapse ◽  
Dna Supercoiling ◽  
Dna Looping ◽  
Microscopy Imaging ◽  
Supercoiled Dna ◽  
Force Microscopy ◽  
Processing Enzymes ◽  
Dna Loop

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, e.g., the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping is stimulated by positive supercoiled DNA where it preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects all nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

scholarly journals Resolving the data asynchronicity in high-speed atomic force microscopy measurement via the Kalman Smoother

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shintaroh Kubo ◽  
Suguru Kato ◽  
Kazuyuki Nakamura ◽  
Noriyuki Kodera ◽  
Shoji Takada
Keyword(s):  
Atomic Force Microscopy ◽  
Kalman Filter ◽  
Single Molecule ◽  
High Speed ◽  
Ground Truth ◽  
Bayesian Filtering ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Kalman Smoother ◽  
Atomic Force

Abstract High-speed atomic force microscopy (HS-AFM) is a scanning probe microscopy that can capture structural dynamics of biomolecules in real time at single molecule level near physiological condition. Albeit much improvement, while scanning one frame of HS-AFM movies, biomolecules often change their conformations largely. Thus, the obtained frame images can be hampered by the time-difference, the asynchronicity, in the data acquisition. Here, to resolve this data asynchronicity in the HS-AFM movie, we developed Kalman filter and smoother methods, some of the sequential Bayesian filtering approaches. The Kalman filter/smoother methods use alternative steps of a short time-propagation by a linear dynamical system and a correction by the likelihood of AFM data acquired pixel by pixel. We first tested the method using a toy model of a diffusing cone, showing that the Kalman smoother method outperforms to reproduce the ground-truth movie. We then applied the Kalman smoother to a synthetic movie for conformational change dynamics of a motor protein, i.e., dynein, confirming the superiority of the Kalman smoother. Finally, we applied the Kalman smoother to two real HS-AFM movies, FlhAC and centralspindlin, reducing distortion and noise in the AFM movies. The method is general and can be applied to any HS-AFM movies.

scholarly journals Multiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Rohit Agarwal ◽  
Karl E. Duderstadt
Keyword(s):  
Single Molecule ◽  
Rare Events ◽  
Dynamic Properties ◽  
Magnetic Tweezers ◽  
Drug Induced ◽  
Single Molecule Level ◽  
Dna Break ◽  
Reaction Cycles ◽  
Enzymatic Pathways ◽  
Routine Imaging

Abstract The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.

Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level

2003 ◽  
Vol 81 (3) ◽  
pp. 151-159 ◽  
Author(s):  
Jordanka Zlatanova ◽  
Sanford H Leuba
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Biological Macromolecules ◽  
High Definition ◽  
Single Molecule Level ◽  
Applied Tension ◽  
Sensitive Tool ◽  
Different Types ◽  
Distinct Features ◽  
Insight Into

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion; understanding the mechanical properties of these molecules helps us understand how they function in the cell. In this review, we summarize the application of magnetic tweezers (MT) to the study of DNA behavior at the single-molecule level. MT can be conveniently used to stretch DNA and introduce controlled levels of superhelicity into the molecule and to follow to a high definition the action of different types of topoisomerases. Its potential for chromatin studies is also enormous, and we will briefly present our first chromatin results.Key words: single-molecules, chromatin, topoisomerases, magnetic tweezers, force.

scholarly journals Mobility of nucleosomes is regulated by their binding partners

2022 ◽  
Author(s):  
Daniel P Melters ◽  
Keir C Neuman ◽  
Tatini Rakshit ◽  
Yamini Dalal
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
High Speed ◽  
Diffusion Constant ◽  
Chromatin Accessibility ◽  
Chromatin Dynamics ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force

Chromatin accessibility is modulated in a variety of ways, both to create open and closed chromatin states which are critical for eukaryotic gene regulation. At the mechanistic single molecule level, how accessibility is regulated remains a fundamental question in the field. Here, we use single molecule tracking by high-speed atomic force microscopy to investigate this question using chromatin arrays and extend our findings into the nucleus. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and observed that the essential kinetochore protein CENP-C reduces the diffusion constant of CENP-A nucleosomes and the linker H1.5 protein restricts H3 nucleosome mobility. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. These data suggest a model in which inner kinetochore proteins are critically involved in modulating chromatin accessibility and consequently, noncoding transcription at human centromeres.

scholarly journals Supercoiling DNA optically

2019 ◽  
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J.L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Torque Control ◽  
Biological Interactions ◽  
Supercoiled Dna ◽  
Dna Structures ◽  
Single Molecule Level ◽  
Open Questions ◽  
Cellular Dna ◽  
Transcription And Replication

AbstractCellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures.1,2,3 For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic,4,5,6 angular-optical,7,8,9 micro-pipette,10 and magneto-optical tweezers.11 However, in order to address many open questions, there is a growing need for new techniques that can combine rapid torque control with both spatial manipulation and fluorescence microscopy. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. Supercoiled DNA formed in this way is amenable to fast buffer exchange, and can be interrogated with both force spectroscopy and fluorescence imaging of the whole DNA. We establish this method as a powerful platform to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

scholarly journals A flow extension tethered particle motion assay for single-molecule proteolysis

2019 ◽  
Author(s):  
Andrew A. Drabek ◽  
Joseph J. Loparo ◽  
Stephen C. Blacklow
Keyword(s):  
Single Molecule ◽  
Magnetic Force ◽  
Magnetic Tweezers ◽  
Signaling Proteins ◽  
Mechanical Tension ◽  
Tobacco Etch Virus Protease ◽  
Single Molecule Level ◽  
Wide Range ◽  
Tethered Particle Motion

AbstractRegulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

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