scholarly journals In silico reconstitution of DNA replication. Lessons from single-molecule imaging and cryo-tomography applied to single-particle cryo-EM

2022 ◽  
Vol 72 ◽  
pp. 279-286
Author(s):  
Julia F. Greiwe ◽  
Giulia Zanetti ◽  
Thomas C.R. Miller ◽  
Alessandro Costa
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Single Particle ◽  
In Silico ◽  
Single Molecule Imaging

scholarly journals Caught in the act: structural dynamics of replication origin activation and fork progression

2020 ◽  
Vol 48 (3) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jacob S. Lewis ◽  
Alessandro Costa
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Single Particle ◽  
Replication Origin ◽  
Origin Activation ◽  
Recent Advances ◽  
Eukaryotic Dna ◽  
New Challenges

This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro. We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.

scholarly journals DNA Manipulation and Single-Molecule Imaging

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1050
Author(s):  
Shunsuke Takahashi ◽  
Masahiko Oshige ◽  
Shinji Katsura
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Dna Molecules ◽  
Dna Manipulation ◽  
Single Dna Molecules ◽  
Biological Studies ◽  
Physical Form ◽  
Almost All

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

scholarly journals Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

2020 ◽  
Author(s):  
Stefan Niekamp ◽  
Nico Stuurman ◽  
Nan Zhang ◽  
Ronald D. Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
In Silico ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Motor Protein ◽  
Single Molecule Imaging ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule binding domains (MTBD) that step along the track. Here, we simultaneously tracked two MTBDs and one AAA ring of a single dynein, as it undergoes hundreds of steps with nanometer precision using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take different sized steps. This variability in the movement between AAA ring and MTBD results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

scholarly journals Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

2021 ◽  
Vol 118 (31) ◽  
pp. e2101391118
Author(s):  
Stefan Niekamp ◽  
Nico Stuurman ◽  
Nan Zhang ◽  
Ronald D. Vale
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
In Silico ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Motor Protein ◽  
Single Molecule Imaging ◽  
Microtubule Binding ◽  
Binding Domains ◽  
Single Molecule Studies

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

scholarly journals In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair

FEBS Letters ◽  
2014 ◽  
Vol 588 (19) ◽  
pp. 3585-3594 ◽  
Author(s):  
Mathew Stracy ◽  
Stephan Uphoff ◽  
Federico Garza de Leon ◽  
Achillefs N. Kapanidis
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Bacterial Dna ◽  
Bacterial Dna Replication

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

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