scholarly journals Real-time detection of condensin-driven DNA compaction reveals a multistep binding mechanism

2017 ◽  
Author(s):  
Jorine M. Eeftens ◽  
Shveta Bisht ◽  
Jacob Kerssemakers ◽  
Christian H. Haering ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Electrostatic Interactions ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Magnetic Tweezers ◽  
Condensed State ◽  
Binding Modes ◽  
Dna Molecules ◽  
Dna Compaction

ABSTRACTCondensin, a conserved member of the SMC protein family of ring-shaped multi-subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single-molecule magnetic tweezers to measure, in real-time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction proceeds in large (~200nm) steps, driving DNA molecules into a fully condensed state against forces of up to 2pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt-insensitive and for subsequent compaction. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Madeline M Keenen ◽  
David Brown ◽  
Lucy D Brennan ◽  
Roman Renger ◽  
Harrison Khoo ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Genome Organization ◽  
Stable Phase ◽  
Dna Molecules ◽  
Weakly Bound ◽  
Dna Compaction ◽  
Polymer Properties ◽  
Molecular Scale ◽  
Hp1 Proteins

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

scholarly journals NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

PLoS ONE ◽  
2012 ◽  
Vol 7 (9) ◽  
pp. e46306 ◽  
Author(s):  
Rifka Vlijm ◽  
Jeremy S. J. Smitshuijzen ◽  
Alexandra Lusser ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Nucleosome Assembly

scholarly journals Human cohesin compacts DNA by loop extrusion

Science ◽  
2019 ◽  
Vol 366 (6471) ◽  
pp. 1345-1349 ◽  
Author(s):  
Yoori Kim ◽  
Zhubing Shi ◽  
Hongshan Zhang ◽  
Ilya J. Finkelstein ◽  
Hongtao Yu
Keyword(s):  
Adenosine Triphosphate ◽  
Single Molecule ◽  
Adenosine Triphosphatase ◽  
Direct Evidence ◽  
Atp Hydrolysis ◽  
Average Rate ◽  
Molecular Machine ◽  
Single Molecule Imaging ◽  
Dna Loops ◽  
Dna Compaction

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

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 A Horizontal Magnetic Tweezers for Studying Single DNA Molecules and DNA-Binding Proteins

Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 4781
Author(s):  
Roberto Fabian ◽  
Santosh Gaire ◽  
Christopher Tyson ◽  
Raghabendra Adhikari ◽  
Ian Pegg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Magnetic Tweezers ◽  
Low Noise ◽  
Core Histone ◽  
Dna Molecules ◽  
Single Dna Molecules ◽  
Core Histones ◽  
Noise Data ◽  
Single Molecule Studies ◽  
Report Data

We report data from single molecule studies on the interaction between single DNA molecules and core histones using custom-designed horizontal magnetic tweezers. The DNA-core histone complexes were formed using λ-DNA tethers, core histones, and NAP1 and were exposed to forces ranging from ~2 pN to ~74 pN. During the assembly events, we observed the length of the DNA decrease in approximate integer multiples of ~50 nm, suggesting the binding of the histone octamers to the DNA tether. During the mechanically induced disassembly events, we observed disruption lengths in approximate integer multiples of ~50 nm, suggesting the unbinding of one or more octamers from the DNA tether. We also observed histone octamer unbinding events at forces as low as ~2 pN. Our horizontal magnetic tweezers yielded high-resolution, low-noise data on force-mediated DNA-core histone assembly and disassembly processes.

scholarly journals Rescuing Replication from Barriers: Mechanistic Insights from Single-Molecule Studies

2019 ◽  
Vol 39 (10) ◽  
Author(s):  
Bo Sun
Keyword(s):  
Real Time ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Replication Fork ◽  
Magnetic Tweezers ◽  
Origin Of Replication ◽  
Replication Fork Progression ◽  
Nucleotide Incorporation ◽  
Single Molecule Studies ◽  
Replication Failure

ABSTRACT To prevent replication failure due to fork barriers, several mechanisms have evolved to restart arrested forks independent of the origin of replication. Our understanding of these mechanisms that underlie replication reactivation has been aided through unique dynamic perspectives offered by single-molecule techniques. These techniques, such as optical tweezers, magnetic tweezers, and fluorescence-based methods, allow researchers to monitor the unwinding of DNA by helicase, nucleotide incorporation during polymerase synthesis, and replication fork progression in real time. In addition, they offer the ability to distinguish DNA intermediates after obstacles to replication at high spatial and temporal resolutions, providing new insights into the replication reactivation mechanisms. These and other highlights of single-molecule techniques and remarkable studies on the recovery of the replication fork from barriers will be discussed in this review.

scholarly journals NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

2013 ◽  
Vol 104 (2) ◽  
pp. 38a-39a
Author(s):  
Rifka Vlijm ◽  
Jeremy S.J. Smitshuijzen ◽  
Alexandra Lusser ◽  
Cees Dekker
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Magnetic Tweezers ◽  
Nucleosome Assembly

scholarly journals Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

2019 ◽  
Author(s):  
T. B. Brouwer ◽  
N. Hermans ◽  
J. van Noort
Keyword(s):  
Diffraction Pattern ◽  
Real Time ◽  
Single Molecule ◽  
Fourier Transforms ◽  
Magnetic Tweezers ◽  
Fast Fourier Transforms ◽  
Tracking Algorithm ◽  
Wide Field ◽  
3D Tracking ◽  
Chromatin Fibers

AbstractMany single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in a z-range of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required.SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10’s of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

scholarly journals HP1 proteins compact DNA into mechanically and positionally stable phase separated domains

2020 ◽  
Author(s):  
Madeline M. Keenen ◽  
David Brown ◽  
Lucy D. Brennan ◽  
Roman Renger ◽  
Harrison Khoo ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Genome Organization ◽  
Stable Phase ◽  
Dna Molecules ◽  
Weakly Bound ◽  
Dna Compaction ◽  
Polymer Properties ◽  
Molecular Scale ◽  
Hp1 Proteins

In mammals HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

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