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 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.

In situ monitoring of single molecule binding reactions with time-lapse atomic force microscopy on functionalized DNA origami

Nanoscale ◽  
2011 ◽  
Vol 3 (6) ◽  
pp. 2481 ◽  
Author(s):  
Na Wu ◽  
Xingfei Zhou ◽  
Daniel M. Czajkowsky ◽  
Ming Ye ◽  
Dongdong Zeng ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Single Molecule ◽  
Time Lapse ◽  
Dna Origami ◽  
In Situ Monitoring ◽  
Force Microscopy ◽  
Atomic Force

DNA-loop Formation on Nucleosomes Shown by in situ Scanning Force Microscopy of Supercoiled DNA

2005 ◽  
Vol 345 (4) ◽  
pp. 695-706 ◽  
Author(s):  
Malte Bussiek ◽  
Katalin Tóth ◽  
Nathalie Brun ◽  
Jörg Langowski
Keyword(s):  
Scanning Force Microscopy ◽  
Loop Formation ◽  
Supercoiled Dna ◽  
Force Microscopy ◽  
Dna Loop ◽  
Scanning Force

scholarly journals Negative DNA supercoiling tunes protein-mediated looping

2021 ◽  
Author(s):  
Yan Yan ◽  
Wenxuan Xu ◽  
Sandip Kumar ◽  
Alexander Zhang ◽  
Fenfei Leng ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Supercoiling ◽  
Lac Repressor ◽  
Emergent Behavior ◽  
Dna Looping ◽  
Inherent Feature ◽  
Loop Dynamics ◽  
Negative Supercoiling ◽  
Deterministic Behavior ◽  
Fundamental Mechanism

Protein-mediated DNA looping is a fundamental mechanism of gene regulation. Such loops occur stochastically, and a calibrated response to environmental stimuli would seem to require more deterministic behavior, so experiments were preformed to determine whether additional proteins and/or DNA supercoiling might be definitive. In experiments on DNA looping mediated by the Escherichia coli lac repressor protein, increasing compaction by the nucleoid-associated protein, HU, progressively increased the average looping probability for an ensemble of single molecules. Despite this trend, the looping probabilities associated with individual molecules ranged from 0 to 100 throughout the titration, and observations of a single molecule for an hour or longer were required to observe the statistical looping behavior of the ensemble, ergodicity. Increased negative supercoiling also increased the looping probability for an ensemble of molecules, but the looping probabilities of individual molecules more closely resembled the ensemble average. Furthermore, supercoiling accelerated the loop dynamics such that in as little as twelve minutes of observation most molecules exhibited the looping probability of the ensemble. Notably, this is within the timescale of the doubling time of the bacterium. DNA supercoiling, an inherent feature of genomes across kingdoms, appears to be a fundamental determinant of time-constrained, emergent behavior in otherwise random molecular activity.

scholarly journals AFM images of open and collapsed states of yeast condensin suggest a scrunching model for DNA loop extrusion

2019 ◽  
Author(s):  
Je-Kyung Ryu ◽  
Allard J. Katan ◽  
Eli O. van der Sluis ◽  
Thomas Wisse ◽  
Ralph de Groot ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Molecular Motor ◽  
Neck Size ◽  
Force Microscopy ◽  
Double Stranded Dna ◽  
Atomic Force ◽  
Condensin Complex ◽  
Dna Loop ◽  
Structural Maintenance Of Chromosome ◽  
Collapsed States

SUMMARYStructural Maintenance of Chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex, which compacts DNA during mitosis, was recently shown to be a molecular motor that extrudes large loops of DNA. The mechanism of this unique motor, which takes large steps along DNA at low ATP consumption, remains elusive however. Here, we use Atomic Force Microscopy (AFM) to visualize the structure of yeast condensin and condensin-DNA complexes. Condensin is found to exhibit mainly open ‘O’ shapes and collapsed ‘B’ shapes, and it cycles dynamically between these two states over time. Condensin binds double-stranded DNA via a HEAT subunit and, surprisingly, also via the hinge domain. On extruded DNA loops, we observe a single condensin complex at the loop stem, where the neck size of the DNA loop correlates with the width of the condensin complex. Our results suggest that condensin extrudes DNA by a fast cyclic switching of its conformation between O and B shapes, consistent with a scrunching model.

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 Single molecule mechanics of the kinesin neck

2009 ◽  
Vol 106 (17) ◽  
pp. 6992-6997 ◽  
Author(s):  
Thomas Bornschlögl ◽  
Günther Woehlke ◽  
Matthias Rief
Keyword(s):  
Single Molecule ◽  
Leucine Zipper ◽  
Structural Integrity ◽  
Mechanical Stability ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Force Microscopy ◽  
Atomic Force ◽  
Kinetics Of ◽  
High Mechanical Stability

Structural integrity as well as mechanical stability of the parts of a molecular motor are crucial for its function. In this study, we used high-resolution force spectroscopy by atomic force microscopy to investigate the force-dependent opening kinetics of the neck coiled coil of Kinesin-1 from Drosophila melanogaster. We find that even though the overall thermodynamic stability of the neck is low, the average opening force of the coiled coil is >11 pN when stretched with pulling velocities >150 nm/s. These high unzipping forces ensure structural integrity during motor motion. The high mechanical stability is achieved through a very narrow N-terminal unfolding barrier if compared with a conventional leucine zipper. The experimentally mapped mechanical unzipping profile allows direct assignment of distinct mechanical stabilities to the different coiled-coil subunits. The coiled-coil sequence seems to be tuned in an optimal way to ensure both mechanical stability as well as motor regulation through charged residues.

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