LIVIVO - Search results -

Search results

Result 1 - 10 of total 104

Search options

Article ; Online: De novo fabrication of custom-sequence plasmids for the synthesis of long DNA constructs with extrahelical features.

Ramírez Montero, Daniel / Liu, Zhaowei / Dekker, Nynke H

2023 Volume 123, Issue 1, Page(s) 31–41

Abstract: DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the ... ...

Abstract	DNA constructs for single-molecule experiments often require specific sequences and/or extrahelical/noncanonical structures to study DNA-processing mechanisms. The precise introduction of such structures requires extensive control of the sequence of the initial DNA substrate. A commonly used substrate in the synthesis of DNA constructs is plasmid DNA. Nevertheless, the controlled introduction of specific sequences and extrahelical/noncanonical structures into plasmids often requires several rounds of cloning on pre-existing plasmids whose sequence one cannot fully control. Here, we describe a simple and efficient way to synthesize 10.1-kb plasmids de novo using synthetic gBlocks that provides full control of the sequence. Using these plasmids, we developed a 1.5-day protocol to assemble 10.1-kb linear DNA constructs with end and internal modifications. As a proof of principle, we synthesize two different DNA constructs with biotinylated ends and one or two internal 3' single-stranded DNA flaps, characterize them using single-molecule force and fluorescence spectroscopy, and functionally validate them by showing that the eukaryotic replicative helicase Cdc45/Mcm2-7/GINS (CMG) binds the 3' single-stranded DNA flap and translocates in the expected direction. We anticipate that our approach can be used to synthesize custom-sequence DNA constructs for a variety of force and fluorescence single-molecule spectroscopy experiments to interrogate DNA replication, DNA repair, and transcription.
MeSH term(s)	DNA, Single-Stranded ; Cell Cycle Proteins/metabolism ; DNA/chemistry ; DNA Replication ; Plasmids/genetics
Chemical Substances	DNA, Single-Stranded ; Cell Cycle Proteins ; DNA (9007-49-2)
Language	English
Publishing date	2023-11-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2023.11.008
Database	MEDical Literature Analysis and Retrieval System OnLINE

Full text online

Accessible to users with ZB MED library card

In stock of ZB MED Cologne/Königswinter

Zs.A 235: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MO 2: Show issues

Order via subito

This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

Details ▾

Article ; Online: Characterizing single-molecule dynamics of viral RNA-dependent RNA polymerases with multiplexed magnetic tweezers.

Kuijpers, Louis / van Laar, Theo / Janissen, Richard / Dekker, Nynke H

STAR protocols

2022 Volume 3, Issue 3, Page(s) 101606

Abstract: Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of ... ...

Abstract	Multiplexed single-molecule magnetic tweezers (MT) have recently been employed to probe the RNA synthesis dynamics of RNA-dependent RNA polymerases (RdRp). Here, we present a protocol for simultaneously probing the RNA synthesis dynamics of hundreds of single polymerases with MT. We describe the preparation of a dsRNA construct for probing single RdRp kinetics. We then detail the measurement of RdRp RNA synthesis kinetics using MT. The protocol is suitable for high-throughput probing of RdRp-targeting antiviral compounds for mechanistic function and efficacy. For complete details on the use and execution of this protocol, please refer to Janissen et al. (2021).
MeSH term(s)	Antiviral Agents ; Kinetics ; Magnetic Phenomena ; RNA, Double-Stranded ; RNA-Dependent RNA Polymerase
Chemical Substances	Antiviral Agents ; RNA, Double-Stranded ; RNA-Dependent RNA Polymerase (EC 2.7.7.48)
Language	English
Publishing date	2022-08-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2022.101606
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Inter-library loan at ZB MED

Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

Article ; Online: Automated cell counting for Trypan blue-stained cell cultures using machine learning.

Kuijpers, Louis / van Veen, Edo / van der Pol, Leo A / Dekker, Nynke H

PloS one

2023 Volume 18, Issue 11, Page(s) e0291625

Abstract: Cell counting is a vital practice in the maintenance and manipulation of cell cultures. It is a crucial aspect of assessing cell viability and determining proliferation rates, which are integral to maintaining the health and functionality of a culture. ... ...

Abstract	Cell counting is a vital practice in the maintenance and manipulation of cell cultures. It is a crucial aspect of assessing cell viability and determining proliferation rates, which are integral to maintaining the health and functionality of a culture. Additionally, it is critical for establishing the time of infection in bioreactors and monitoring cell culture response to targeted infection over time. However, when cell counting is performed manually, the time involved can become substantial, particularly when multiple cultures need to be handled in parallel. Automated cell counters, which enable significant time reduction, are commercially available but remain relatively expensive. Here, we present a machine learning (ML) model based on YOLOv4 that is able to perform cell counts with a high accuracy (>95%) for Trypan blue-stained insect cells. Images of two distinctly different cell lines, Trichoplusia ni (High FiveTM; Hi5 cells) and Spodoptera frugiperda (Sf9), were used for training, validation, and testing of the model. The ML model yielded F1 scores of 0.97 and 0.96 for alive and dead cells, respectively, which represents a substantially improved performance over that of other cell counters. Furthermore, the ML model is versatile, as an F1 score of 0.96 was also obtained on images of Trypan blue-stained human embryonic kidney (HEK) cells that the model had not been trained on. Our implementation of the ML model comes with a straightforward user interface and can image in batches, which makes it highly suitable for the evaluation of multiple parallel cultures (e.g. in Design of Experiments). Overall, this approach for accurate classification of cells provides a fast, bias-free alternative to manual counting.
MeSH term(s)	Animals ; Humans ; Trypan Blue ; Cell Count/methods ; Cell Culture Techniques ; Cell Line ; Spodoptera
Chemical Substances	Trypan Blue (I2ZWO3LS3M)
Language	English
Publishing date	2023-11-28
Publishing country	United States
Document type	Journal Article
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0291625
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Article ; Online: High-throughput single-molecule experiments reveal heterogeneity, state switching, and three interconnected pause states in transcription.

Janissen, Richard / Eslami-Mossallam, Behrouz / Artsimovitch, Irina / Depken, Martin / Dekker, Nynke H

Cell reports

2022 Volume 39, Issue 4, Page(s) 110749

Abstract: Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding ...

Abstract	Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examine the Escherichia coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identify, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We identify two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlies elongation-rate switching, and variations in RNA cleavage rates in long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.
MeSH term(s)	DNA-Directed RNA Polymerases/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; RNA, Bacterial ; Transcription, Genetic
Chemical Substances	RNA, Bacterial ; DNA-Directed RNA Polymerases (EC 2.7.7.6)
Language	English
Publishing date	2022-04-25
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2022.110749
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Article ; Online: A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint.

Sánchez, Humberto / Liu, Zhaowei / van Veen, Edo / van Laar, Theo / Diffley, John F X / Dekker, Nynke H

Nature communications

2023 Volume 14, Issue 1, Page(s) 6735

Abstract: Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ...

Abstract	Chromatin replication involves the assembly and activity of the replisome within the nucleosomal landscape. At the core of the replisome is the Mcm2-7 complex (MCM), which is loaded onto DNA after binding to the Origin Recognition Complex (ORC). In yeast, ORC is a dynamic protein that diffuses rapidly along DNA, unless halted by origin recognition sequences. However, less is known about the dynamics of ORC proteins in the presence of nucleosomes and attendant consequences for MCM loading. To address this, we harnessed an in vitro single-molecule approach to interrogate a chromatinized origin of replication. We find that ORC binds the origin of replication with similar efficiency independently of whether the origin is chromatinized, despite ORC mobility being reduced by the presence of nucleosomes. Recruitment of MCM also proceeds efficiently on a chromatinized origin, but subsequent movement of MCM away from the origin is severely constrained. These findings suggest that chromatinized origins in yeast are essential for the local retention of MCM, which may facilitate subsequent assembly of the replisome.
MeSH term(s)	Origin Recognition Complex/genetics ; Origin Recognition Complex/metabolism ; Nucleosomes ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Cell Cycle Proteins/metabolism ; DNA/metabolism ; DNA Replication ; Minichromosome Maintenance Proteins/genetics ; Minichromosome Maintenance Proteins/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Replication Origin
Chemical Substances	Origin Recognition Complex ; Nucleosomes ; Cell Cycle Proteins ; DNA (9007-49-2) ; Minichromosome Maintenance Proteins (EC 3.6.4.12) ; Saccharomyces cerevisiae Proteins
Language	English
Publishing date	2023-10-23
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-42524-8
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Article ; Online: Nucleotide binding halts diffusion of the eukaryotic replicative helicase during activation.

Ramírez Montero, Daniel / Sánchez, Humberto / van Veen, Edo / van Laar, Theo / Solano, Belén / Diffley, John F X / Dekker, Nynke H

Nature communications

2023 Volume 14, Issue 1, Page(s) 2082

Abstract: The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is ... ...

Abstract	The eukaryotic replicative helicase CMG centrally orchestrates the replisome and leads the way at the front of replication forks. Understanding the motion of CMG on the DNA is therefore key to our understanding of DNA replication. In vivo, CMG is assembled and activated through a cell-cycle-regulated mechanism involving 36 polypeptides that has been reconstituted from purified proteins in ensemble biochemical studies. Conversely, single-molecule studies of CMG motion have thus far relied on pre-formed CMG assembled through an unknown mechanism upon overexpression of individual constituents. Here, we report the activation of CMG fully reconstituted from purified yeast proteins and the quantification of its motion at the single-molecule level. We observe that CMG can move on DNA in two ways: by unidirectional translocation and by diffusion. We demonstrate that CMG preferentially exhibits unidirectional translocation in the presence of ATP, whereas it preferentially exhibits diffusive motion in the absence of ATP. We also demonstrate that nucleotide binding halts diffusive CMG independently of DNA melting. Taken together, our findings support a mechanism by which nucleotide binding allows newly assembled CMG to engage with the DNA within its central channel, halting its diffusion and facilitating the initial DNA melting required to initiate DNA replication.
MeSH term(s)	Eukaryota/metabolism ; Nucleotides ; DNA Replication ; DNA Helicases/metabolism ; DNA/metabolism ; Adenosine Triphosphate/metabolism
Chemical Substances	Nucleotides ; DNA Helicases (EC 3.6.4.-) ; DNA (9007-49-2) ; Adenosine Triphosphate (8L70Q75FXE)
Language	English
Publishing date	2023-04-14
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-37093-9
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Article: A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy.

Liu, Zhaowei / van Veen, Edo / Sánchez, Humberto / Solano, Belén / Palmero Moya, Francisco J / McCluskey, Kaley A / Ramírez Montero, Daniel / van Laar, Theo / Dekker, Nynke H

ACS photonics

2024 Volume 11, Issue 4, Page(s) 1592–1603

Abstract: Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual- ...

Abstract	Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.
Language	English
Publishing date	2024-03-18
Publishing country	United States
Document type	Journal Article
ISSN	2330-4022
ISSN	2330-4022
DOI	10.1021/acsphotonics.3c01739
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Inter-library loan at ZB MED

Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

Article ; Online: DNA Sequence Is a Major Determinant of Tetrasome Dynamics.

Ordu, Orkide / Lusser, Alexandra / Dekker, Nynke H

Biophysical journal

2019 Volume 117, Issue 11, Page(s) 2217–2227

Abstract: Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4) ...

Abstract	Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)
MeSH term(s)	Animals ; Base Sequence ; Chromosomes/drug effects ; Chromosomes/genetics ; DNA/chemistry ; DNA/genetics ; Drosophila/genetics ; Kinetics ; Models, Molecular ; Nucleic Acid Conformation ; Salts/pharmacology ; Thermodynamics
Chemical Substances	Salts ; DNA (9007-49-2)
Language	English
Publishing date	2019-08-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2019.07.055
Database	MEDical Literature Analysis and Retrieval System OnLINE

Full text online

Accessible to users with ZB MED library card

In stock of ZB MED Cologne/Königswinter

Zs.A 235: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MO 2: Show issues

Order via subito

Details ▾

Article ; Online: Chromatin fibers stabilize nucleosomes under torsional stress.

Kaczmarczyk, Artur / Meng, He / Ordu, Orkide / Noort, John van / Dekker, Nynke H

Nature communications

2020 Volume 11, Issue 1, Page(s) 126

Abstract: Torsional stress generated during DNA replication and transcription has been suggested to facilitate nucleosome unwrapping and thereby the progression of polymerases. However, the propagation of twist in condensed chromatin remains yet unresolved. Here, ... ...

Abstract	Torsional stress generated during DNA replication and transcription has been suggested to facilitate nucleosome unwrapping and thereby the progression of polymerases. However, the propagation of twist in condensed chromatin remains yet unresolved. Here, we measure how force and torque impact chromatin fibers with a nucleosome repeat length of 167 and 197. We find that both types of fibers fold into a left-handed superhelix that can be stabilized by positive torsion. We observe that the structural changes induced by twist were reversible, indicating that chromatin has a large degree of elasticity. Our direct measurements of torque confirmed the hypothesis of chromatin fibers as a twist buffer. Using a statistical mechanics-based torsional spring model, we extracted values of the chromatin twist modulus and the linking number per stacked nucleosome that were in good agreement with values measured here experimentally. Overall, our findings indicate that the supercoiling generated by DNA-processing enzymes, predicted by the twin-supercoiled domain model, can be largely accommodated by the higher-order structure of chromatin.
MeSH term(s)	Chromatin/chemistry ; Chromatin/genetics ; Chromatin/metabolism ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; DNA Replication ; Elasticity ; Humans ; Nucleic Acid Conformation ; Nucleosomes/chemistry ; Nucleosomes/genetics ; Nucleosomes/metabolism ; Torque
Chemical Substances	Chromatin ; Nucleosomes ; DNA (9007-49-2)
Language	English
Publishing date	2020-01-08
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-019-13891-y
Database	MEDical Literature Analysis and Retrieval System OnLINE

Order via subito

Article ; Online: Modification of the histone tetramer at the H3-H3 interface impacts tetrasome conformations and dynamics.

Ordu, Orkide / Kremser, Leopold / Lusser, Alexandra / Dekker, Nynke H

The Journal of chemical physics

2018 Volume 148, Issue 12, Page(s) 123323

Abstract: Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential ... ...

Abstract	Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential genomic processes such as replication, transcription, and repair. The assembly of nucleosomes obeys a precise pathway in which tetramers of histones H3 and H4 bind to the DNA first to form tetrasomes, and two dimers of histones H2A and H2B are subsequently incorporated to complete the complex. As viable intermediates, we previously showed that tetrasomes can spontaneously flip between a left-handed and right-handed conformation of DNA-wrapping. To pinpoint the underlying mechanism, here we investigated the role of the H3-H3 interface for tetramer flexibility in the flipping process at the single-molecule level. Using freely orbiting magnetic tweezers, we studied the assembly and structural dynamics of individual tetrasomes modified at the cysteines close to this interaction interface by iodoacetamide (IA) in real time. While such modification did not affect the structural properties of the tetrasomes, it caused a 3-fold change in their flipping kinetics. The results indicate that the IA-modification enhances the conformational plasticity of tetrasomes. Our findings suggest that subnucleosomal dynamics may be employed by chromatin as an intrinsic and adjustable mechanism to regulate DNA supercoiling.
MeSH term(s)	Histones/chemistry ; Histones/classification ; Iodoacetamide/chemistry ; Molecular Conformation
Chemical Substances	Histones ; Iodoacetamide (ZRH8M27S79)
Language	English
Publishing date	2018-03-15
Publishing country	United States
Document type	Journal Article
ZDB-ID	3113-6
ISSN	1089-7690 ; 0021-9606
ISSN (online)	1089-7690
ISSN	0021-9606
DOI	10.1063/1.5009100
Database	MEDical Literature Analysis and Retrieval System OnLINE

In stock of ZB MED Bonn / Germany

Z 4' 49/16: Show issues

Order via subito

Inter-library loan at ZB MED

Your chosen title can be delivered directly to ZB MED Cologne location if you are registered as a user at ZB MED Cologne.

Details ▾
- See ZB MED holdings
- Order with fees

To top

Full text online

More links

Kategorien

In stock of ZB MED Cologne/Königswinter

Order via subito

More links

Kategorien

Order via subito

Inter-library loan at ZB MED

More links

Kategorien

Order via subito

More links

Kategorien

Order via subito

More links

Kategorien

Order via subito

More links

Kategorien

Order via subito

More links

Kategorien

Order via subito

Inter-library loan at ZB MED

Full text online

More links

Kategorien

In stock of ZB MED Cologne/Königswinter

Order via subito

More links

Kategorien

Order via subito

More links

Kategorien

In stock of ZB MED Bonn / Germany

Order via subito

Inter-library loan at ZB MED