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Liu, Z. (author), van Veen, E.N.W. (author), Sanchez González, H. (author), Solano Hermosilla, B.P. (author), Palmero Moya, F.J.P.M. (author), Mc Cluskey, K.A. (author), Ramirez Montero, D.F. (author), van Laar, T. (author), Dekker, N.H. (author)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...journal article 2024
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- Dekker, N.H. (author) journal article 2023
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Rouillon, Clément (author), Eckhardt, Bruna V. (author), Kollenstart, Leonie (author), Gruss, Fabian (author), Verkennis, Alexander E.E. (author), Rondeel, Inge (author), van Laar, T. (author), Albanese, Pascal (author), Dekker, N.H. (author)During every cell cycle, both the genome and the associated chromatin must be accurately replicated. Chromatin Assembly Factor-1 (CAF-1) is a key regulator of chromatin replication, but how CAF-1 functions in relation to the DNA replication machinery is unknown. Here, we reveal that this crosstalk differs between the leading and lagging...journal article 2023
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Kuijpers, L.C. (author), van Veen, E.N.W. (author), Van der Pol, Leo (author), Dekker, N.H. (author)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...journal article 2023
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Mc Cluskey, K.A. (author), Dekker, N.H. (author)TIRF (Total Internal Reflection Fluorescence) microscopy is a powerful tool for measuring the intra- and intermolecular dynamics of fluorescently-labeled single molecules. As TIRF measurements move to more complex biological systems with more fluorescent probes, the multi-band-pass dichroic that separates excitation from emission becomes...journal article 2023
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Ramirez Montero, D.F. (author), Sanchez González, H. (author), van Veen, E.N.W. (author), van Laar, T. (author), Solano Hermosilla, B.P. (author), Diffley, John F.X. (author), Dekker, N.H. (author)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...journal article 2023
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Ramirez Montero, D.F. (author), Liu, Z. (author), Dekker, N.H. (author)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...journal article 2023
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Fan, D. (author), Cnossen, J.P. (author), Hung, S. (author), Kromm, D. (author), Dekker, N.H. (author), Verbiest, G.J. (author), Smith, C.S. (author)To better understand the interactions between biological molecules, a high optical resolution in all three dimensions is crucial. The intrinsically lower axial resolution of microscopes however, is a limiting factor in fluorescence imaging, correspondingly in fluorescence based single molecule localization microscopy (SMLM). Here, we present...journal article 2023
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Sanchez González, H. (author), Liu, Z. (author), van Veen, E.N.W. (author), van Laar, T. (author), Diffley, John F.X. (author), Dekker, N.H. (author)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...journal article 2023
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Janissen, R. (author), Eslami Mossallam, B. (author), Artsimovitch, Irina (author), Depken, S.M. (author), Dekker, N.H. (author)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...journal article 2022
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Kuijpers, L.C. (author), van Laar, T. (author), Janissen, R. (author), Dekker, N.H. (author)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...journal article 2022
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Mc Cluskey, K.A. (author), van Veen, E.N.W. (author), Cnossen, J.P. (author), Wesselink, W.J. (author), Asscher, F.M. (author), Smith, C.S. (author), Dekker, N.H. (author)Accurate image alignment is critical in multicolor single-molecule fluorescence microscopy. Global alignment using affine transformations leaves residual errors due to the nonlinearity of the distortions, which decreases the effective field of view. Subsequent local refinement demands either large amounts of reference data and processing time...journal article 2021
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Janissen, R. (author), Woodman, Andrew (author), Shengjuler, Djoshkun (author), Vallet, Thomas (author), Lee, Kuo Ming (author), Kuijpers, L.C. (author), Moustafa, Ibrahim M. (author), Fitzgerald, Fiona (author), Dekker, N.H. (author)Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics...journal article 2021
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Sanchez González, H. (author), Mc Cluskey, K.A. (author), van Laar, T. (author), van Veen, E.N.W. (author), Asscher, F.M. (author), Solano Hermosilla, B.P. (author), Diffley, John F.X. (author), Dekker, N.H. (author)DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the...journal article 2021
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Tang, Y. (author), Ha, S. (author), Begou, Thomas (author), Lumeau, Julien (author), Urbach, Paul (author), Dekker, N.H. (author), Adam, A.J.L. (author)The ability to apply force and torque directly to micro- and nanoscale particles in optical traps has a wide range of applications. While full control of both force and torque in three dimensions has been realized using top-down fabrication of rod-shaped particles composed of birefringent crystalline materials, widespread usage of such...journal article 2020
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Kaczmarczyk, A.P. (author), Meng, He (author), Ordu, O. (author), Noort, John van (author), Dekker, N.H. (author)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...journal article 2020
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Ha, S. (author), Tang, Y. (author), van Oene, M.M. (author), Janissen, R. (author), Dries, R.M. (author), Solano Hermosilla, B.P. (author), Adam, A.J.L. (author), Dekker, N.H. (author)Optical trapping of (sub)micron-sized particles is broadly employed in nanoscience and engineering. The materials commonly employed for these particles, however, have physical properties that limit the transfer of linear or angular momentum (or both). This reduces the magnitude of forces and torques, and the spatiotemporal resolution,...journal article 2019
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Ordu, O. (author), Lusser, Alexandra (author), Dekker, N.H. (author)Eukaryotic genomes are hierarchically organized into protein-DNA assemblies for compaction into the nucleus. Nucleosomes, with the (H3-H4)<sub>2</sub> tetrasome as a likely intermediate, are highly dynamic in nature by way of several different mechanisms. We have recently shown that tetrasomes spontaneously change the direction of their DNA...journal article 2019
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Lee, Kuo Ming (author), Gong, Yu Nong (author), Hsieh, Tzu Hsuan (author), Woodman, Andrew (author), Dekker, N.H. (author), Cameron, Craig E. (author), Shih, Shin Ru (author)Enterovirus A71 (EV-A71) is an important nonpolio enterovirus that causes severe neurological complications. In 1998, Taiwan experienced an EV-A71 outbreak that caused 78 deaths. Since then, periodic epidemics of EV-A71 associated with newly emerging strains have occurred. Several of these strains are known to be recombinant; however, how...journal article 2018
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van Oene, M.M. (author), Ha, S. (author), Jager, T. (author), Lee, M. (author), Pedaci, F. (author), Lipfert, J. (author), Dekker, N.H. (author)Single-molecule manipulation techniques have provided unprecedented insights into the structure, function, interactions, and mechanical properties of biological macromolecules. Recently, the single-molecule toolbox has been expanded by techniques that enable measurements of rotation and torque, such as the optical torque wrench (OTW) and...journal article 2018