Richard Janissen | Delft University of Technology (original) (raw)

Papers by Richard Janissen

ABSTRACTTranscription-coupled supercoiling of DNA is a key factor in chromosome compaction and th... more ABSTRACTTranscription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model within vitroreal-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried ou...

Nature, Apr 19, 2023

To obtain insights into how CTCF controls cohesin, we developed in vitro assays in which CTCF-coh... more To obtain insights into how CTCF controls cohesin, we developed in vitro assays in which CTCF-cohesin interactions can be visualized on DNA at the single-molecule level in real time. We first analysed how CTCF finds its DNA consensus sequence. Consistent with previous

Biophysical Journal, 2014

We report a surface passivation method for single-molecule studies that can decrease non-specific... more We report a surface passivation method for single-molecule studies that can decrease non-specific binding of biomolecules by at least 10-fold as compared to the widely used polyethylene glycol (PEG) surface. As demonstrated for a variety of biological systems, the new surface does not perturb the behavior and activities of biomolecules. Reduction in preparation time and cost is another major advantage. The reported approach can replace the PEG protocol and expand the reach of the powerful single-molecule experimental tools.

Cell Reports, Apr 1, 2022

High-throughput single-molecule experiments reveal heterogeneity, state switching, and three inte... more High-throughput single-molecule experiments reveal heterogeneity, state switching, and three interconnected pause states in transcription

Biophysical Journal, Feb 1, 2023

bioRxiv (Cold Spring Harbor Laboratory), Nov 4, 2020

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ... more The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-bindinginduced engagement of the hinge and the globular domain of the SMC complex.

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2021

Highlights d RdRp ''backtracked'' state is an intermediate for template switching d Intra-and int... more Highlights d RdRp ''backtracked'' state is an intermediate for template switching d Intra-and intermolecular template switching use the same ''backtracked'' intermediate d Pyrazine-carboxamide nucleotides dysregulate recombination, increasing defective genomes d Alternative mechanistic class and target of antiviral nucleotides are revealed

Biophysical Journal, 2015

PIFE, an acronym that stands for ''Protein Induced Fluorescence Enhancement'', is a term that has... more PIFE, an acronym that stands for ''Protein Induced Fluorescence Enhancement'', is a term that has been coined to describe the enhancement of fluorescence intensity that the dye Cy3 experiences in the proximity of a protein. The approach has been used to study dynamic aspects of a large number of DNAand RNA-protein interactions at the single molecule level. We and others have hypothesized that the phenomenon results from a restriction in the photoisomerization deactivation pathway of the dye. Here, we present the results of a detailed spectroscopic study that aims to characterize PIFE at the molecular level. We used time-resolved fluorescence, fluorescence anisotropy and transient spectroscopy to fully characterize the deactivation pathways of the dye on DNA when next to a protein. Our results allowed us to confirm our hypothesis that the enhancement in fluorescence correlates with a decreased yield of photoisomer.

Journal of the American Chemical Society, Dec 12, 2022

Mechanophores are powerful molecular tools used to track bond rupture and characterize mechanical... more Mechanophores are powerful molecular tools used to track bond rupture and characterize mechanical damage in polymers. The majority of mechanophores are known to respond to external stresses, and we report in this study the first precedent of a mechanochemical response to internal, residual stresses that accumulate during polymer vitrification. While internal stress is intrinsic to polymers that can form solids, we demonstrate that it can dramatically affect the mechanochemistry of spiropyran probes and alter their intramolecular isomerization barriers by up to 70 kJ mol–1. This new behavior of spiropyrans (SPs) enables their application for analysis of internal stresses distribution and their mechanochemical characterization on the molecular level. Spectroscopy and imaging based on SP mechanochemistry showed high topological sensitivity and allowed us to discern different levels of internal stress impacting various locations along the polymer chain. The nature of the developed technique allows for wide-field imaging of stress heterogeneities in polymer samples of irregular shapes and dimensions, making it feasible to directly observe molecular-level manifestations of mechanical stresses that accompany the formation of a vast number of solid polymers.

Nano Letters, Jun 30, 2016

Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric sub... more Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cellsurface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex-vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues. Single cell bacterial adhesion to a surface is the first, and critical, step to originate a biofilm. After a community of attached microorganisms is formed, it embeds itself in a matrix of hydrated extracellular polymeric substances (EPS), which includes a variety of biomolecules, such as DNA, oligopeptides, proteins, lipids and lipopolysaccharides. 1-5 EPS secretion represents an important factor for the motile-to sessile-stage transition during bacterial cell adhesion. Furthermore, specific extracellular components often facilitate adhesion by reducing the energy barrier formed between approaching surfaces, 6-8 in a process which depends mostly on bacteria-host adaptation mechanisms. 9-10 Thus, it is important to probe the specific dependence of bacterial cell adhesion on surface chemical composition and the corresponding molecular mechanisms. EPS-mediated adhesion is also noteworthy of similar studies; after initial cell attachment, the secreted EPS eventually accumulates, leading to changes in biofilm stiffness and elasticity. In that sense, a quantitative analysis of cell-surface interaction mechanisms may eventually lead to new strategies for biofilm eradication, a serious technological challenge in several and very different areas, such as health care and agriculture. 1,3,5,11 Further complexity to the scenario of bacterial biofilm infection arises from the ability of many species to modulate cell adhesiveness, and hence host colonization, in response to changing environmental conditions. 12 In fact, bacterial cells respond to many cues from the surface such as wettability 13,14 , chemical composition 15 , compliance 16,17 and

Social Science Research Network, 2021

bioRxiv (Cold Spring Harbor Laboratory), Jan 9, 2021

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptiv... more The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse pathogens filamentous cells have been observed during biofilm formation, with function yet to be elucidated. To identify prior hypothesized quorum sensing as trigger of such cell morphogenesis, spatially controlled cell adhesion is pivotal. Here, we demonstrate highly-selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters using these patterns provided evidence of quorum sensing governing filamentous cell formation. While cell morphogenesis is induced by cell cluster density, filamentous cell growth is oriented towards neighboring cell clusters and distance-dependent; large interconnected cell clusters create

Scientific Reports, Apr 20, 2015

In most bacteria, chromosome segregation is driven by the ParABSsystem where the CTPase protein P... more In most bacteria, chromosome segregation is driven by the ParABSsystem where the CTPase protein ParB loads at theparSsite to trigger the formation of a large partition complex. Here, we presentin vitrostudies of the partition complex forBacillus subtilisParB, using single-molecule fluorescence microscopy and AFM imaging to show that transient ParB-ParB bridges are essential for forming DNA condensates. Molecular Dynamics simulations confirm that condensation occurs abruptly at a critical concentration of ParB and show that multimerization is a prerequisite for forming the partition complex. Magnetic tweezer force spectroscopy on mutant ParB proteins demonstrates that CTP hydrolysis at the N-terminal domain is essential for DNA condensation. Finally, we show that transcribing RNA polymerases can steadily traverse the ParB-DNA partition complex. These findings uncover how ParB forms a stable yet dynamic partition complex for chromosome segregation that induces DNA condensation and seg...

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the D... more In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CTCF generates topologically associating domains (TADs)2-4 that play key roles in gene regulation and recombination during development and disease1,5-8. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unknown9. To address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, as well as to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted, but unexpectedly is dependent on DNA tension. Moreover, CTCF regulates cohesin’s loop extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-me...

Mechanophores are powerful molecular tools used in polymers to track bond rupture and characteriz... more Mechanophores are powerful molecular tools used in polymers to track bond rupture and characterize mechanical damage. The majority of mechanophores are known to respond to external stresses and in this study we report the first precedent of a mechanochemical response to internal, residual stresses that accumulate during polymer vitrification. While internal stress is intrinsic to polymers that can form solids, we demonstrate that it can dramatically affect the mechanochemistry of spyropyran probes and alter their intramolecular isomerization barriers by up to 70 kJ·mol-1. This new behavior of spiropyrans enables their application for analysis of internal stresses and their mechanochemical characterization on the molecular level. Spectroscopy and imaging based on spiropyran mechanochemistry showed high topological sensitivity and allowed to discern different levels of internal stress in various locations along the polymer chain. The nature of the developed technique allows for wide f...

The research object was the characterization of two enterovirus RNA-dependent RNA polymerases usi... more The research object was the characterization of two enterovirus RNA-dependent RNA polymerases using multiplexed magnetic tweezers, to investigate snap-back synthesis and recombination. Presented here are <i>in vitro</i> measurements of the bead position during RNA replication. Bead position measurements were taken for 2 hours a constant pulling force of 25 pN at 24C at a camera acquisition rate of 50 Hz. Traces were cropped from the start until the end of the change in z-position, which was converted to nucleotides.

Recent advances in optical tweezers have enabled the direct manipulation and measurement of optic... more Recent advances in optical tweezers have enabled the direct manipulation and measurement of optical torque using light spin momentum transferred to trapped birefringent particles. This powerful technique, termed Optical Torque Wrench (OTW), relies on trapping of quartz (SiO2) microcylinders which have become a standard and convenient probe for single-molecule studies. Here, we explore an alternative photonic probe based on rutile (TiO2) which has almost thirty-fold larger birefringence compared to quartz particles. By employing this promising material to fabricate rutile nanocylinders whose sizes can be easily tuned, we significantly enhance the accessible range of optical torques and angular frequencies in the OTW. In future, these novel photonic probes will allow us to study not only slowly processing torque-generating biological systems, as the genome processing machinery, but also fast rotating motors, including ATP-synthase and the bacterial flagellar motor.

SSRN Electronic Journal, 2020

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ... more The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATPdependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ~50 nm protein complex. Here, using magnetic tweezers, we resolve single steps in loop extrusion by individual yeast condensins. Step sizes range between 20-45 nm at forces of 1.0-0.2 pN, respectively, comparable to the complex size. The large steps show that condensin reels in DNA in very sizeable amounts, up to ~600 bp per extrusion step, consistent with the non-stretched DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss the findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATPbinding-induced engagement of the hinge and the globular domain of the SMC complex.

Nature, Apr 19, 2023

Biophysical Journal, 2014

Cell Reports, Apr 1, 2022

Biophysical Journal, Feb 1, 2023

bioRxiv (Cold Spring Harbor Laboratory), Nov 4, 2020

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2021

Biophysical Journal, 2015

Journal of the American Chemical Society, Dec 12, 2022

Nano Letters, Jun 30, 2016

Social Science Research Network, 2021

bioRxiv (Cold Spring Harbor Laboratory), Jan 9, 2021

Scientific Reports, Apr 20, 2015

SSRN Electronic Journal, 2020

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ... more The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATPdependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ~50 nm protein complex. Here, using magnetic tweezers, we resolve single steps in loop extrusion by individual yeast condensins. Step sizes range between 20-45 nm at forces of 1.0-0.2 pN, respectively, comparable to the complex size. The large steps show that condensin reels in DNA in very sizeable amounts, up to ~600 bp per extrusion step, consistent with the non-stretched DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss the findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATPbinding-induced engagement of the hinge and the globular domain of the SMC complex.