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Dr. rer. nat. Richard Janissen, PhD

Forschungsleiter

Principal Investigator - Group Leader Research Director Bioengineering BITZ Transformation Lab Bavarian Innovation Transformation Center Oberschneiding


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Bericht/Report
  • I. Davidson
  • R. Barth
  • M. Zaczek
  • J. van der Torre
  • W. Tang
  • K. Nagasaka
  • Richard Janissen
  • J. Kerssemakers
  • G. Wutz
  • C. Dekker
  • J.-M. Peters

CTCF is a DNA-tension-dependent barrier to cohesin-mediated DNA loop extrusion.

(2022)

Zeitschriftenartikel
  • Richard Janissen
  • L. Oberbarnscheidt
  • F. Oesterhelt

Optimized straight forward procedure for covalent surface immobilization of different biomolecules for single molecule applications.

In: Colloids and Surfaces. B, Biointerfaces (vol. 71) , pg. 200-7

(2009)

DOI: 10.1016/j.colsurfb.2009.02.011

Covalent chemisorption of biomolecules to surfaces with high density and low unspecific background is prerequisite for most optical and mechanical single molecule experiments and accordingly, many recipes have been developed. However, new establishment of the surface functionalization process in the lab usually is still difficult and time consuming due to the complex procedures containing many pitfalls. Therefore, based on the known recipes, we developed and optimized a simple straight forward protocol. We demonstrated it resulting in a high density of the coupled biomolecules, homogeneous surfaces and a low unspecific background when binding nucleic acids, peptides and proteins. The protocol was optimized for borosilicate cover glasses and silicon nitride atomic force microscope cantilevers commonly used in single molecule experiments and takes advantage of commonly used chemicals. It consists of only four steps, silanol group generation, amination, grafting of poly(ethylene glycol) to the surface and biomolecule coupling. All individual steps were optimized comparing different variations partially described in the literature. Finally, a detailed description is provided which allows avoiding most sources of contamination, often being a main hurdle on the way to single molecule experiments.
Zeitschriftenartikel
  • L. Oberbarnscheidt
  • Richard Janissen
  • F. Oesterhelt

Direct and model free calculation of force-dependent dissociation rates from force spectroscopic data.

In: Biophysical Journal (vol. 97) , pg. L19-21

(2009)

DOI: 10.1016/j.bpj.2009.08.015

Force spectroscopy allows testing the free energy landscapes of molecular interactions. Usually, the dependency of the most probable rupture force on the force rate or the shape of the rupture force histogram is fitted with different models that contain approximations and basic assumptions. We present a simple and model free approach to extract the force-dependent dissociation rates directly from the force curve data. Simulations show that the dissociation rates at any force are given directly by the ratio of the number of detected rupture events to the time this force was acting on the bond. To calculate these total times of acting forces, all force curve data points of all curves measured are taken into account, which significantly increases the amount of information which is considered for data analysis compared to other methods. Moreover, by providing force-dependent dissociation rates this method allows direct testing and validating of any energy landscape model.
Zeitschriftenartikel
  • L. Oberbarnscheidt
  • Richard Janissen
  • S. Martell
  • M. Engelhard
  • F. Oesterhelt

Single-molecule force spectroscopy measures structural changes induced by light activation and transducer binding in sensory rhodopsin II.

In: Journal of Molecular Biology (vol. 394) , pg. 383-90

(2009)

DOI: 10.1016/j.jmb.2009.07.083

Microbial rhodopsins are a family of seven-helical transmembrane proteins containing retinal as chromophore. Sensory rhodopsin II (SRII) triggers two very different responses upon light excitation, depending on the presence or the absence of its cognate transducer HtrII: Whereas light activation of the NpSRII/NpHtrII complex activates a signalling cascade that initiates the photophobic response, NpSRII alone acts as a proton pump. Using single-molecule force spectroscopy, we analysed the stability of NpSRII and its complex with the transducer in the dark and under illumination. By improving force spectroscopic data analysis, we were able to reveal the localisation of occurring forces within the protein chain with a resolution of about six amino acids. Distinct regions in helices G and F were affected differently, depending on the experimental conditions. The results are generally in line with previous data on the molecular stability of NpSRII. Interestingly, new interaction sites were identified upon light activation, whose functional importance is discussed in detail.
Zeitschriftenartikel
  • Richard Janissen
  • F. Oesterhelt

Directed deposition of single molecules on surfaces.

In: Journal of Nanoscience and Nanotechnology (vol. 10) , pg. 5328-32

(2010)

DOI: 10.1166/jnn.2010.2382

Scanning probe microscopy-based techniques can address and manipulate individual molecules. This makes it possible to use them for building nanostructures by assembling single molecules. Recently the formation of surface structures by positioning single molecules with the Atomic Force Microscope (AFM) was demonstrated on an irreversible delivery process. This inherits the drawback, that the transfer has to occur between differently functionalized surfaces and allows no proofreading of the built structures. Here we demonstrate a procedure for directed deposition of single DNA molecules, which intrinsically allows a reversible positioning. This method uses specific interactions between complementary DNA oligonucleotides for symmetric coupling of the transport molecules to the support and AFM tip, respectively. Thus, it allows for a simple "drag-and-drop" procedure, which relies on the statistical breakage of the molecular interaction under a force load. In addition, the delivery of the transport molecules was observed in real-time by single-molecule fluorescence microscopy.
Zeitschriftenartikel
  • P. Rothwell
  • W. Allen
  • E. Sisamakis
  • Richard Janissen
  • S. Kalinin
  • S. Felekyan
  • J. Widengren
  • G. Waksman
  • C. Seidel

dNTP-Dependent Conformational Transitions in the Fingers Subdomain of Klentaq1.

In: Biophysical Journal (vol. 100) , pg. 376a

(2011)

DOI: 10.1016/j.bpj.2010.12.2238

DNA polymerases are responsible for the accurate replication of DNA. Kinetic studies indicate the requirement for multiple intermediate enzyme conformations in this process. Structural studies show a large conformational change in the fingers subdomain of DNA polymerase on binding of a correct dNTP. Using single molecule FRET we show that the conformational transition affecting the fingers subdomain also takes place in the apo and DNA-bound forms of the enzyme. In addition a third conformation is observed which is occupied in the presence of dNTP alone, and in the presence of a non base pairing dNTPs. The relative proportions of the identified states are altered dramatically depending on substrate. Binding of the correct nucleotide displaces this equilibrium dramatically towards the closed form, while binding of an incorrect nucleotide favors a more open conformation. The results suggest that the closed state is by design less energetically favored, providing a thermodynamic brake on incorrect nucleotide insertion.
Zeitschriftenartikel
  • M. Toledo
  • Richard Janissen
  • M. Favaro
  • M. Cotta
  • G. Monteiro
  • D. Prazeres
  • A. Souza
  • A. Azzoni

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery.

In: Journal of Controlled Release : Official journal of the Controlled Release Society (vol. 159) , pg. 222-31

(2012)

DOI: 10.1016/j.jconrel.2012.01.011

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies.
Zeitschriftenartikel
  • A. Moreau
  • Richard Janissen
  • C. Santos
  • L. Peroni
  • D. Stach-Machado
  • A. Souza
  • A. Souza
  • M. Cotta

Highly-sensitive and label-free indium phosphide biosensor for early phytopathogen diagnosis.

In: Biosensors & Bioelectronics (vol. 36) , pg. 62-8

(2012)

DOI: 10.1016/j.bios.2012.03.038

The development of highly-sensitive and label-free operating semiconductor-based, biomaterial detecting sensors has important applications in areas such as environmental science, biomedical research and medical diagnostics. In the present study, we developed an Indium Phosphide (InP) semiconductor-based resistive biosensor using the change of its electronic properties upon biomaterial adsorption as sensing element. To detect biomaterial at low concentrations, the procedure of functionalization and covalent biomolecule immobilization was also optimized to guarantee high molecule density and high reproducibility which are prerequisite for reliable results. The characterization, such as biomolecular conjugation efficiency, detection concentration limits, receptor:ligand specificity and concentration detection range was analyzed by using three different biological systems: i) synthetic dsDNA and two phytopathogenic diseases, ii) the severe CB-form of Citrus Tristeza Virus (CTV) and iii) Xylella fastidiosa, both causing great economic loss worldwide. The experimental results show a sensitivity of 1 pM for specific ssDNA detection and about 2 nM for the specific detection of surface proteins of CTV and X. fastidiosa phytopathogens. A brief comparison with other semiconductor based biosensors and other methodological approaches is discussed and confirms the high sensitivity and reproducibility of our InP based biosensor which could be suitable for reliable early infection diagnosis in environmental and life sciences.
Zeitschriftenartikel
  • M. Jäger
  • C. Böge
  • Richard Janissen
  • D. Rohrbeck
  • T. Hülsen
  • S. Lensing-Höhn
  • R. Krauspe
  • M. Herten

Osteoblastic potency of bone marrow cells cultivated on functionalized biometals with cyclic RGD-peptide.

In: Journal of Biomedical Materials Research. Part A (vol. 101) , pg. 2905-14

(2013)

DOI: 10.1002/jbm.a.34590

The fixation of cementless endoprostheses requires excellent fixation at the bone implant interface. Although the surface structures of these implants are designed to promote osteoblastic differentiation, poor bone quality may prevent or delay osseointegration. There is evidence that RGD peptides known as recognition motifs for various integrins, promote cellular adhesion, influence cellular proliferation, and differentiation of local cells. In this study, five different metal surfaces were analyzed: Sandblasted (TiSa) and polished (TiPol) Ti6Al4V, porocoated (CCPor) and polished (CCPol) cobalt chrome and polished stainless steel (SS) were coated by ethanol amine and poly(ethylene glycol) to attach covalently RGD peptides. Human mesenchymal stromal cells of healthy donors were cultivated onto prior functionalized metal surfaces for 14 days without osteogenic stimulation. Cell proliferation and differentiation were quantitatively evaluated for native (I), NaOH pre-activated (II), NaOH pre-activated, and PEG-coated (III) as well as for RGD (IV) coated surfaces. The RGD immobilization efficiency was analyzed by epi-fluorescence spectroscopy, cell morphology was documented by light and scanning electron microscopy. The RGD-binding efficiency was TiSa > TiPol > SS > CCPor > CCPol. RGD coated surfaces showed the highest average cell proliferation on CCPol > SS > CCPor > TiSa ≥ TiPol, whereas cellular differentiation mostly correlated with the observed proliferation results, such as CCPol > TiSa > SS > CCPor > TiPol. Considering statistical analyses (significance level of α = 0.05), the RGD-coating of all biometals in comparison and in respect of their particular controls showed no significant improvement in cellular proliferation and osteoblastic differentiation.
Zeitschriftenartikel
  • G. Lorite
  • Richard Janissen
  • J. Clerici
  • C. Rodrigues
  • J. Tomaz
  • B. Mizaikoff
  • C. Kranz
  • A. Souza
  • M. Cotta

Surface physicochemical properties at the micro and nano length scales: role on bacterial adhesion and Xylella fastidiosa biofilm development.

In: PLoS One (vol. 8) , pg. e75247

(2013)

DOI: 10.1371/journal.pone.0075247

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.
Zeitschriftenartikel
  • Richard Janissen
  • B. Berghuis
  • D. Dulin
  • M. Wink
  • T. van Laar
  • N. Dekker

Invincible DNA tethers: covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments.

In: Nucleic Acids Research (vol. 42) , pg. e137

(2014)

DOI: 10.1093/nar/gku677

Magnetic tweezers are a powerful single-molecule technique that allows real-time quantitative investigation of biomolecular processes under applied force. High pulling forces exceeding tens of picoNewtons may be required, e.g. to probe the force range of proteins that actively transcribe or package the genome. Frequently, however, the application of such forces decreases the sample lifetime, hindering data acquisition. To provide experimentally viable sample lifetimes in the face of high pulling forces, we have designed a novel anchoring strategy for DNA in magnetic tweezers. Our approach, which exploits covalent functionalization based on heterobifunctional poly(ethylene glycol) crosslinkers, allows us to strongly tether DNA while simultaneously suppressing undesirable non-specific adhesion. A complete force and lifetime characterization of these covalently anchored DNA-tethers demonstrates that, compared to more commonly employed anchoring strategies, they withstand 3-fold higher pulling forces (up to 150 pN) and exhibit up to 200-fold higher lifetimes (exceeding 24 h at a constant force of 150 pN). This advance makes it possible to apply the full range of biologically relevant force scales to biomolecular processes, and its straightforward implementation should extend its reach to a multitude of applications in the field of single-molecule force spectroscopy.
Zeitschriftenartikel
  • M. Favaro
  • M. Toledo
  • R. Alves
  • C. Santos
  • L. Beloti
  • Richard Janissen
  • L. La Torre
  • A. Souza
  • A. Azzoni

Development of a non-viral gene delivery vector based on the dynein light chain Rp3 and the TAT peptide.

In: Journal of Biotechnology (vol. 173) , pg. 10-8

(2014)

DOI: 10.1016/j.jbiotec.2014.01.001

Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (∼100nm) and positively charged (+28.6mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000™, but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery.
Zeitschriftenartikel
  • S. Ha
  • M. van Oene
  • Richard Janissen
  • N. Dekker

Fabrication and Surface Functionalization of Highly Birefringent Particles for Optical Torque Wrench.

In: Biophysical Journal (vol. 106) , pg. 393a

(2014)

DOI: 10.1016/j.bpj.2013.11.2220

Conventional optical tweezers are limited by the ability to apply only translational manipulation to probe the diverse biological systems. The recent extension of optical tweezers, i.e., the optical torque wrench (OTW), allows the direct application and measurement of torque. The OTW provides a platform to measure rotational dynamics of biomolecules and motors including the torque-dependence of DNA or DNA-protein interactions, and of powerful machines such as F0F1-ATP-synthase or bacterial flagellar motor. The applicable torque of the OTW is largely dependent on the birefringence of the trapped particle. Quartz (SiO2) is widely used due to its facile fabrication and stability in biological environments. However, the birefringence of quartz is limited to fully investigate the torque-speed relationships of diverse biological systems so we explored more highly birefringent crystals such as rutile (TiO2) and vanadate (YVO4). Developing novel fabrication protocol is required because the particle fabrication from these alternative crystals is not as straightforward as quartz. For example, Cr was used as a mask for dry etching cylinders from rutile with SF6/CH4 processing gases and it resulted in etching selectivity of 1:28 which is higher than electron beam resist mask with selectivity of 1:1.3. To promote the selective attachment of the particle to target biological system, a submicron-sized protrusion can be fabricated on one end of a cylindrical particle and functionalized with specific ligand biomolecules. Alkoxysilane based surface crosslinkers, such as APTES, that are commonly used to bind biomolecules to solid supports, tend to polymerize and result in inhomogeneous surface coatings. To obtain uniform, dense, and reproducible surface coating for nanoscale structures, we explored the use of cyclic aza-silanes as alternative crosslinkers for biomolecule attachment.
Zeitschriftenartikel
  • Richard Janissen
  • B. Berghuis
  • O. Ordu
  • M. Wink
  • D. Dulin
  • J. Cnossen
  • N. Dekker

Single Molecule Studies of DNA-Binding Proteins: Development of New Covalent DNA Anchoring Techniques for the Study of Rupture Forces of Replication Blocks.

In: Biophysical Journal (vol. 106) , pg. 394a

(2014)

DOI: 10.1016/j.bpj.2013.11.2224

Magnetic Tweezers (MT) are a powerful technique to investigate the dynamics and kinetics of biomechanical processes in-vitro on a single-molecule level. In certain cases, it is necessary to apply unusually high pulling forces for the mechanochemical characterization of biomolecule structures and complexes, such as protein-nucleic acid complexes, DNA and RNA conformational overstretching conformation transition phenomena, or the unfolding of polymers and proteins at high resolution. To achieve high pulling forces using MT, we have redesigned the anchoring strategy of biomolecules to the surface and magnetic bead, employing a bottom-up covalent chemisorption procedure. The development covers several aspects such as surface passivation to avoid aspecific physisorption, flexible and interchangeable covalent binding strategies using inert poly(ethylene glycol) linkers, and the characterization of chemical stability of tethered dsDNA sample constructs and overall coating density. Using this novel approach, dsDNA constructs have reproducibly and reliably withstood pulling forces of > 140 pN over long observation times. We have applied our novel anchoring strategy to anchor DNA hairpins containing a single replication termination (ter) sequence. In E.coli, the DNA binding protein Tus binds to ter sites and is known to bring approaching replication forks to a halt by blocking strand separation. Upon opening our DNA hairpin in the presence of counter-helicase Tus, we mimicked replisome activity, and strand separation was demonstrably blocked. However, the barrier imposed was larger than our non-covalent anchoring method could withstand; it was impossible to ‘break the lock’. Employing our new method, we are able to apply forces > 140 pN to the Tus:ter complex, allowing us to characterize the energy landscape of this system.
Zeitschriftenartikel
  • S. Ha
  • Y. Ussembayev
  • Richard Janissen
  • M. van Oene
  • N. Dekker

Fabrication and Surface Functionalization of Highly Birefringent Rutile Particles for Trapping in an Optical Torque Wrench.

In: Biophysical Journal (vol. 108) , pg. 337a

(2015)

DOI: 10.1016/j.bpj.2014.11.1838

The optical torque wrench (OTW) allows the direct application and measurement of torque on biomolecules, such as DNA or DNA-protein complexes, or rotary motors like the F0F1-ATP-synthase or the bacterial flagellar motor. The applicable torque of the OTW is a function of the size and birefringence of the particle. Quartz has proven a convenient material, but its quite low birefringence limits full investigation of torque-speed relationships of diverse biological systems. In contrast, rutile exhibits a much higher birefringence - exceeding that of quartz by a factor of 30 - but its utilization has been infrequent because of the difficulties in optical trapping and fabrication. To enhance the applicability of the OTW, we have improved both the design and fabrication of cylindrical rutile particles. We have employed finite element method calculations to determine the optimal dimension of stably trappable rutile cylinders. To obtain rutile cylinders with the optimal dimensions, we developed a protocol for full control of size and sidewall angle. In our fabrication protocol, a chromium etch mask provides increased resistance to dry etching and allows the fabrication of structures with both high aspect ratio and anisotropy. Also, the sidewall angle of cylinders can be readily tuned by adjusting a single process parameter, namely the oxygen flow rate during dry etching. The fabricated cylinders were characterized in the OTW setup to reveal their linear and angular trapping properties. The fabrication process is compatible with common chemical functionalization procedures and permits covalent biomolecule attachment. To enhance biomolecule coverage, we used ethanolamine and poly(ethylene glycol) as biomolecular crosslinkers to obtain homogenous and dense coatings. Our recent results, in which we use functionalized, trapped rutile cylinders to study single biomolecules and motor proteins, will be presented.
Zeitschriftenartikel
  • Richard Janissen
  • D. Murillo
  • B. Niza
  • P. Sahoo
  • M. Nobrega
  • C. Cesar
  • M. Temperini
  • H. Carvalho
  • A. Souza
  • M. Cotta

Spatiotemporal distribution of different extracellular polymeric substances and filamentation mediate Xylella fastidiosa adhesion and biofilm formation.

In: Scientific Reports (Nature Publishing Group) (vol. 5) , pg. 9856

(2015)

DOI: 10.1038/srep09856

Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.
Zeitschriftenartikel
  • C. Santos
  • Richard Janissen
  • M. Toledo
  • L. Beloti
  • A. Azzoni
  • M. Cotta
  • A. Souza

Characterization of the TolB-Pal trans-envelope complex from Xylella fastidiosa reveals a dynamic and coordinated protein expression profile during the biofilm development process.

In: Biochimica et Biophysica Acta (vol. 1854) , pg. 1372-81

(2015)

DOI: 10.1016/j.bbapap.2015.05.018

The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development.
Zeitschriftenartikel
  • E. Ostrofet
  • S. Ha
  • Richard Janissen
  • T. van Laar
  • N. Dekker

Stretched, Oriented DNA Arrays (SODA) for Fluorescence Based Single-Molecule Experiments in Complex Environment.

In: Biophysical Journal (vol. 108) , pg. 321a

(2015)

DOI: 10.1016/j.bpj.2014.11.1747

All biochemical and biophysical processes that support cellular activity take place in complex, crowded environments. Up to 30% of the weight of a cell consists of proteins, DNA and other large biological macromolecules. Consequently, 1-dimensional protein motions along DNA while replication or transcription have to be studied and understood in the context of a DNA molecule that is not naked, but instead bound by a wide variety of obstacles - roadblocks. Inspired by previous, pioneering work on DNA curtains, we used the intrinsic propriety of some macromolecules and polymers to create self-assembled, organized structures, adapted for visualization using TIRF microscopy of interactions between genome processing enzymes and roadblocks in crowded environment.
Zeitschriftenartikel
  • B. Berghuis
  • D. Dulin
  • Z.-Q. Xu
  • T. van Laar
  • B. Cross
  • Richard Janissen
  • S. Jergic
  • N. Dixon
  • M. Depken
  • N. Dekker

Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier.

In: Nature Chemical Biology (vol. 11) , pg. 579-85

(2015)

DOI: 10.1038/nchembio.1857

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.
Zeitschriftenartikel
  • Y. Ussembayev
  • S. Ha
  • Richard Janissen
  • M. van Oene
  • N. Dekker

Trapping of Highly Birefringent Rutile Nanocylinders in the Optical Torque Wrench.

In: Biophysical Journal (vol. 110) , pg. 499a

(2016)

DOI: 10.1016/j.bpj.2015.11.2668

The optical torque wrench (OTW) is a powerful technique to measure the torsional properties of different biomolecules, including DNA, DNA-processing protein complexes and rotary motors. To date, quartz has proven to be a convenient birefringent material out of which to synthesize the micron-sized particles essential for this technique. However, the relatively low birefringence of quartz, which limits the maximal torque that can be applied in OTW, hampers the study of certain biological systems. A more attractive material is rutile, which has a thirty-fold higher birefringence. To date, however, the application of rutile in the trapping has been restricted due to its high refractive index, which results in low trapping efficiency. Here, we have employed finite element method calculations to determine the optimal dimensions of sub-micron-sized rutile cylinders for tight stable optical trapping. Using these calculations as a guideline, we have designed and developed a nanofabrication protocol that allows us to produce rutile cylinders with the desired sizes at high yield. We have characterized the fabricated cylinders in the OTW setup and quantified both their linear and angular trapping properties. In addition, we demonstrate full translational and rotational control of these functionalized cylinders tethered to individual DNA molecules for use in single-molecule applications.
Zeitschriftenartikel
  • S. Ha
  • Richard Janissen
  • Y. Ussembayev
  • M. Oene
  • B. Solano
  • N. Dekker

Tunable top-down fabrication and functional surface coating of single-crystal titanium dioxide nanostructures and nanoparticles.

In: Nanoscale (vol. 8) , pg. 10739-48

(2016)

DOI: 10.1039/c6nr00898d

Titanium dioxide (TiO2) is a key component of diverse optical and electronic applications that exploit its exceptional material properties. In particular, the use of TiO2 in its single-crystalline phase can offer substantial advantages over its amorphous and polycrystalline phases for existing and yet-to-be-developed applications. However, the implementation of single-crystal TiO2 has been hampered by challenges in its fabrication and subsequent surface functionalization. Here, we introduce a novel top-down approach that allows for batch fabrication of uniform high-aspect-ratio single-crystal TiO2 nanostructures with targeted sidewall profiles. We complement our fabrication approach with a functionalization strategy that achieves dense, uniform, and area-selective coating with a variety of biomolecules. This allows us to fabricate single-crystal rutile TiO2 nanocylinders tethered with individual DNA molecules for use as force- and torque-transducers in an optical torque wrench. These developments provide the means for increased exploitation of the superior material properties of single-crystal TiO2 at the nanoscale.
Zeitschriftenartikel
  • E. Ostrofet
  • S. Ha
  • Richard Janissen
  • T. van Laar
  • N. Dekker

Stretched Oriented DNA Arrays (SODA) as a Tool for Studying Protein-DNA Interactions.

In: Biophysical Journal (vol. 110) , pg. 166a

(2016)

DOI: 10.1016/j.bpj.2015.11.925

Stretched Oriented DNA Array (SODA) is a platform for visualization and characterization of protein-DNA and protein-protein interactions on a DNA strand at single-molecule level. Single DNA molecules are arranged in a pre-defined manner, stretched and bound to a solid support at both ends near the glass surface. This configuration enables simultaneous visualization of the dynamics of labelled proteins on hundreds of DNA strands using TIRF microscopy. Due to this high throughput, SODA can be used to observe rare events which may occur during genome processing in the presence of roadblocks. Besides, the microfluidic chamber with passivated surface that has been developed shows promise for experiments in a crowded environment. The combination of these advances makes SODA an appropriate tool for addressing questions related to genome processing in crowded environment.
Beitrag in Sammelwerk/Tagungsband
  • Richard Janissen
  • D. Murillo
  • B. Niza
  • P. Sahoo
  • M. Monteiro
  • C. César
  • H. Carvalho
  • A. Souza
  • M. Cotta

Filamentation and spatiotemporal distribution of extracellular polymeric substances: role on X.fastidiosa single cell adhesion and biofilm formation (Conference Presentation).

  • In:
  • D. Nicolau
  • D. Farkas
  • R. Leif

SPIE pg. 17

DOI: 10.1117/12.2213830

(2016)

Zeitschriftenartikel
  • P. Sahoo
  • Richard Janissen
  • M. Monteiro
  • A. Cavalli
  • D. Murillo
  • et al.

Nanowire Arrays as Cell Force Sensors To Investigate Adhesin-Enhanced Holdfast of Single Cell Bacteria and Biofilm Stability.

In: Nano Letters (vol. 16) , pg. 4656-64

(2016)

DOI: 10.1021/acs.nanolett.6b01998

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 cell-surface 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.
Zeitschriftenartikel
  • Richard Janissen
  • P. Sahoo
  • C. Santos
  • A. Da Silva
  • A. Zuben
  • D. Souto
  • et al.

InP Nanowire Biosensor with Tailored Biofunctionalization: Ultrasensitive and Highly Selective Disease Biomarker Detection.

In: Nano Letters (vol. 17) , pg. 5938-5949

(2017)

DOI: 10.1021/acs.nanolett.7b01803

Electrically active field-effect transistors (FET) based biosensors are of paramount importance in life science applications, as they offer direct, fast, and highly sensitive label-free detection capabilities of several biomolecules of specific interest. In this work, we report a detailed investigation on surface functionalization and covalent immobilization of biomarkers using biocompatible ethanolamine and poly(ethylene glycol) derivate coatings, as compared to the conventional approaches using silica monoliths, in order to substantially increase both the sensitivity and molecular selectivity of nanowire-based FET biosensor platforms. Quantitative fluorescence, atomic and Kelvin probe force microscopy allowed detailed investigation of the homogeneity and density of immobilized biomarkers on different biofunctionalized surfaces. Significantly enhanced binding specificity, biomarker density, and target biomolecule capture efficiency were thus achieved for DNA as well as for proteins from pathogens. This optimized functionalization methodology was applied to InP nanowires that due to their low surface recombination rates were used as new active transducers for biosensors. The developed devices provide ultrahigh label-free detection sensitivities ∼1 fM for specific DNA sequences, measured via the net change in device electrical resistance. Similar levels of ultrasensitive detection of ∼6 fM were achieved for a Chagas Disease protein marker (IBMP8-1). The developed InP nanowire biosensor provides thus a qualified tool for detection of the chronic infection stage of this disease, leading to improved diagnosis and control of spread. These methodological developments are expected to substantially enhance the chemical robustness, diagnostic reliability, detection sensitivity, and biomarker selectivity for current and future biosensing devices.
Zeitschriftenartikel
  • Richard Janissen
  • M. Arens
  • N. Vtyurina
  • Z. Rivai
  • N. Sunday
  • B. Eslami-Mossallam
  • A. Gritsenko
  • et al.

Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription.

In: Cell (vol. 174) , pg. 1188-1199.e14

(2018)

DOI: 10.1016/j.cell.2018.06.049

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.
Bericht/Report
  • A. Woodman
  • K.-M. Lee
  • Richard Janissen
  • Y.-N. Gong
  • N. Dekker
  • S.-R. Shih
  • C. Cameron

Predicting Intra- and Intertypic Recombination in Enterovirus 71.

(2018)

Bericht/Report
  • Richard Janissen
  • B. Eslami-Mossallam
  • I. Artsimovitch
  • M. Depken
  • N. Dekker

Accounting for RNA polymerase heterogeneity reveals state switching and two distinct long-lived backtrack states escaping through cleavage.

(2019)

Zeitschriftenartikel
  • A. Woodman
  • K.-M. Lee
  • Richard Janissen
  • Y.-N. Gong
  • N. Dekker
  • S.-R. Shih
  • C. Cameron

Predicting Intraserotypic Recombination in Enterovirus 71.

In: Journal of Virology (vol. 93)

(2019)

DOI: 10.1128/jvi.02057-18

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.
Zeitschriftenartikel
  • S. Ha
  • Y. Tang
  • M. van Oene
  • Richard Janissen
  • R. Dries
  • B. Solano
  • A. Adam
  • N. Dekker

Single-Crystal Rutile TiO2 Nanocylinders are Highly Effective Transducers of Optical Force and Torque.

In: ACS Photonics (vol. 6) , pg. 1255-1265

(2019)

DOI: 10.1021/acsphotonics.9b00220

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, achievable in linear and angular traps. Here, we overcome these limitations through the use of single-crystal rutile TiO2, which has an exceptionally large optical birefringence, a high index of refraction, good chemical stability, and is amenable to geometric control at the nanoscale. We show that rutile TiO2 nanocylinders form powerful joint force and torque transducers in aqueous environments by using only moderate laser powers to apply nN·nm torques at kHz rotational frequencies to tightly trapped particles. In doing so, we demonstrate how rutile TiO2 nanocylinders outperform other materials and offer unprecedented opportunities to expand the control of optical force and torque at the nanoscale.
Bericht/Report
  • J.-K. Ryu
  • S.-H. Rah
  • Richard Janissen
  • J. Kerssemakers
  • A. Bonato
  • D. Michieletto
  • C. Dekker

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events.

(2020)

Bericht/Report
  • Richard Janissen
  • A. Woodman
  • K.-M. Lee
  • I. Moustafa
  • F. Fitzgerald
  • P.-N. Huang
  • L. Kuijpers
  • A. Perkins
  • D. Harki
  • J. Arnold
  • B. Solano
  • S.-R. Shih
  • C. Cameron
  • N. Dekker

Induced copy-back RNA synthesis as a novel therapeutic mechanism against RNA viruses.

(2020)

Zeitschriftenartikel
  • S. Anbumani
  • A. Da Silva
  • I. Carvalho
  • E. Fischer
  • M. Souza E. Silva
  • A. Zuben
  • H. Carvalho
  • A. Souza
  • Richard Janissen
  • M. Cotta

Controlled spatial organization of bacterial growth reveals key role of cell filamentation preceding Xylella fastidiosa biofilm formation.

In: NPJ Biofilms and Microbiomes (vol. 7) , pg. 86

(2021)

DOI: 10.1038/s41522-021-00258-9

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse human and plant pathogens, filamentous cells have been recently observed during biofilm formation, but their functions and triggering mechanisms remain unclear. To experimentally identify the underlying function and hypothesized cell communication triggers of such cell morphogenesis, spatially controlled cell patterning 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 demonstrated that filamentous cell formation depends on cell cluster density, and their ability to interconnect neighboring cell clusters is distance-dependent. This process allows the creation of large interconnected cell clusters that form the structural framework for macroscale biofilms. The addition of diffusible signaling molecules from supernatant extracts provides evidence that cell filamentation is induced by quorum sensing. These findings and our innovative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.
Bericht/Report
  • S. Anbumani
  • A. Da Silva
  • E. Fischer
  • M. Souza e Silva
  • A. Zuben
  • H. Carvalho
  • A. Souza
  • Richard Janissen
  • M. Cotta

Controlled spatial organization of bacterial clusters reveals cell filamentation is vital for Xylella fastidiosa biofilm formation.

(2021)

Zeitschriftenartikel
  • B. Lehner
  • D. Benz
  • S. Moshkalev
  • A. Meyer
  • M. Cotta
  • Richard Janissen

Biocompatible Graphene Oxide Nanosheets Densely Functionalized with Biologically Active Molecules for Biosensing Applications.

In: ACS Applied Nano Materials (vol. 4) , pg. 8334-8342

(2021)

DOI: 10.1021/acsanm.1c01522

Graphene oxide (GO) has immense potential for widespread use in diverse in vitro and in vivo biomedical applications owing to its thermal and chemical resistance, excellent electrical properties and solubility, and high surface-to-volume ratio. However, development of GO-based biological nanocomposites and biosensors has been hampered by its poor intrinsic biocompatibility and difficult covalent biofunctionalization across its lattice. Many studies exploit the strategy of chemically modifying GO by noncovalent and reversible attachment of (bio)molecules or sole covalent biofunctionalization of residual moieties at the lattice edges, resulting in a low coating coverage and a largely bioincompatible composite. Here, we address these problems and present a facile yet powerful method for the covalent biofunctionalization of GO using colamine (CA) and the poly(ethylene glycol) cross-linker that results in a vast improvement in the biomolecular coating density and heterogeneity across the entire GO lattice. We further demonstrate that our biofunctionalized GO with CA as the cross-linker provides superior nonspecific biomolecule adhesion suppression with increased biomarker detection sensitivity in a DNA-biosensing assay compared to the (3-aminopropyl)triethoxysilane cross-linker. Our optimized biofunctionalization method will aid the development of GO-based in situ applications including biosensors, tissue nanocomposites, and drug carriers.
Zeitschriftenartikel
  • J.-K. Ryu
  • S.-H. Rah
  • Richard Janissen
  • J. Kerssemakers
  • A. Bonato
  • D. Michieletto
  • C. Dekker

Condensin extrudes DNA loops in steps up to hundreds of base pairs that are generated by ATP binding events.

In: Nucleic Acids Research (vol. 50) , pg. 820-832

(2022)

DOI: 10.1093/nar/gkab1268

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-binding-induced engagement of the hinge and the globular domain of the SMC complex.
Sonstiges
  • I. Davidson
  • R. Barth
  • M. Zaczek
  • J. van der Torre
  • W. Tang
  • K. Nagasaka
  • Richard Janissen
  • J. Kerssemakers
  • G. Wutz
  • C. Dekker
  • J.-M. Peters

Custom code accompanying Davidson, Barth, Zaczek et al. - CTCF is a DNA-tension-dependent barrier to cohesin-mediated DNA loop extrusion.

Zenodo.

(2022)

Zeitschriftenartikel
  • Richard Janissen
  • G. Filonenko

Mechanochemistry of Spiropyran under Internal Stresses of a Glassy Polymer.

In: Journal of the American Chemical Society (vol. 144) , pg. 23198-23204

(2022)

DOI: 10.1021/jacs.2c11280

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.
Zeitschriftenartikel
  • L. Kuijpers
  • T. van Laar
  • Richard Janissen
  • N. Dekker

Characterizing single-molecule dynamics of viral RNA-dependent RNA polymerases with multiplexed magnetic tweezers.

In: STAR Protocols (vol. 3) , pg. 101606

(2022)

DOI: 10.1016/j.xpro.2022.101606

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).
Zeitschriftenartikel
  • M. Tišma
  • Richard Janissen
  • H. Antar
  • A. Martin-Gonzalez
  • R. Barth
  • T. Beekman
  • J. van der Torre
  • D. Michieletto
  • S. Gruber
  • C. Dekker

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation.

In: Nucleic Acids Research (vol. 51) , pg. 11856-11875

(2023)

DOI: 10.1093/nar/gkad868

In most bacteria, chromosome segregation is driven by the ParABS system where the CTPase protein ParB loads at the parS site to trigger the formation of a large partition complex. Here, we present in vitro studies of the partition complex for Bacillus subtilis ParB, 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 segregation while enabling replication and transcription.
Bericht/Report
  • M. Tišma
  • Richard Janissen
  • H. Antar
  • A. Gonzalez
  • R. Barth
  • T. Beekman
  • J. van der Torre
  • D. Michieletto
  • S. Gruber
  • C. Dekker

Dynamic ParB-DNA interactions initiate and maintain a partition condensate for bacterial chromosome segregation.

(2023)

Bericht/Report
  • Richard Janissen
  • R. Barth
  • M. Polinder
  • J. van der Torre
  • C. Dekker

Single-molecule visualization of twin-supercoiled domains generated during transcription.

(2023)

Zeitschriftenartikel
  • I. Davidson
  • R. Barth
  • M. Zaczek
  • J. van der Torre
  • W. Tang
  • K. Nagasaka
  • Richard Janissen
  • J. Kerssemakers
  • G. Wutz
  • C. Dekker
  • J.-M. Peters

CTCF is a DNA-tension-dependent barrier to cohesin-mediated loop extrusion.

In: Nature (vol. 616) , pg. 822-827

(2023)

DOI: 10.1038/s41586-023-05961-5

In eukaryotes, genomic DNA is extruded into loops by cohesin1. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)2,3 that have important roles in gene regulation and recombination during development and disease1,4-7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear8. Here, 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, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF 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-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.
Bericht/Report
  • A. Martin Gonzalez
  • M. Tišma
  • B. Analikwu
  • A. Barth
  • Richard Janissen
  • H. Antar
  • G. Kemps
  • S. Gruber
  • C. Dekker

DNA supercoiling enhances DNA condensation by ParB proteins.

(2024)

Zeitschriftenartikel
  • Richard Janissen
  • R. Barth
  • M. Polinder
  • J. van der Torre
  • C. Dekker

Single-molecule visualization of twin-supercoiled domains generated during transcription.

In: Nucleic Acids Research (vol. 52) , pg. 1677-1687

(2024)

DOI: 10.1093/nar/gkad1181

Transcription-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 with in vitro real-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 out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.
Zeitschriftenartikel
  • Richard Janissen
  • R. Barth
  • I. Davidson
  • J.-M. Peters
  • C. Dekker

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA loop extrusion step.

In: Science Advances (vol. 10) , pg. eadt1832

(2024)

DOI: 10.1126/sciadv.adt1832

Eukaryotes carry three types of structural maintenance of chromosome (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion (LE). SMCs modulate DNA supercoiling but remains incompletely understood how this is achieved. Using a single-molecule magnetic tweezers assay that directly measures how much twist is induced by individual SMCs in each LE step, we demonstrate that all three SMC complexes induce the same large negative twist (i.e., linking number change [Formula: see text] of ~-0.6 at each LE step) into the extruded loop, independent of step size and DNA tension. Using ATP hydrolysis mutants and nonhydrolyzable ATP analogs, we find that ATP binding is the twist-inducing event during the ATPase cycle, coinciding with the force-generating LE step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-LE mechanism among these SMC complexes.
Bericht/Report
  • I. Davidson
  • R. Barth
  • S. Horn
  • Richard Janissen
  • K. Nagasaka
  • G. Wutz
  • R. Stocsits
  • B. Bauer
  • C. Dekker
  • J.-M. Peters

Cohesin supercoils DNA during loop extrusion.

(2024)

Bericht/Report
  • Richard Janissen
  • R. Barth
  • I. Davidson
  • M. Taschner
  • S. Gruber
  • J.-M. Peters
  • C. Dekker

All eukaryotic SMC proteins induce a twist of -0.6 at each DNA-loop-extrusion step.

(2024)

Zeitschriftenartikel
  • A. Martin-Gonzalez
  • M. Tišma
  • B. Analikwu
  • A. Barth
  • Richard Janissen
  • H. Antar
  • G. Kemps
  • S. Gruber
  • C. Dekker

DNA supercoiling enhances DNA condensation by ParB proteins.

In: Nucleic Acids Research (vol. 52) , pg. 13255-13268

(2024)

DOI: 10.1093/nar/gkae936

The ParABS system plays a critical role in bacterial chromosome segregation. The key component of this system, ParB, loads and spreads along DNA to form a local protein-DNA condensate known as a partition complex. As bacterial chromosomes are heavily supercoiled due to the continuous action of RNA polymerases, topoisomerases and nucleoid-associated proteins, it is important to study the impact of DNA supercoiling on the ParB-DNA partition complex formation. Here, we use an in-vitro single-molecule assay to visualize ParB on supercoiled DNA. Unlike most DNA-binding proteins, individual ParB proteins are found to not pin plectonemes on supercoiled DNA, but freely diffuse along supercoiled DNA. We find that DNA supercoiling enhances ParB-DNA condensation, which initiates at lower ParB concentrations than on DNA that is torsionally relaxed. ParB proteins induce a DNA-protein condensate that strikingly absorbs all supercoiling writhe. Our findings provide mechanistic insights that have important implications for our understanding of bacterial chromosome organization and segregation.