Biology subjects

Janissen, R.

Publications and source records attributed to Janissen, R..

12 recordsLinked to original sources

bioRxiv2025

Two CTCF motifs impede cohesin-mediated DNA loop extrusion

Cohesin extrudes DNA into loops and is positioned along the genome by stalling at CTCF upon encountering its N-terminal region (NTR). The mechanism underlying this stalling, however, is unresolved. Using single-molecule assays that monitor DNA loop extrusion (LE) in the presence of NTR fragments, we identify two amino acid motifs, YDF and KTYQR, that hinder LE. KTYQR is found to fully impede LE activity, while YDF hinders cohesin to complete LE step cycles and converts cohesin into a unidirectional extruder by strengthening the affinity of STAG1 to DNA. We thus identify two distinct NTR motifs that stall LE via different yet synergistic mechanisms, highlighting the multifaceted ways employed by CTCF to modulate LE to shape and regulate genomes. One-Sentence SummaryThe N-terminus of CTCF employs two independent motifs that synergistically stall cohesin-mediated DNA loop extrusion.

biophysics↗

bioRxiv2024

DNA supercoiling enhances DNA condensation by ParB proteins

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, gyrases, 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.

biophysics↗

bioRxiv2024

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

Eukaryotes carry three types of Structural Maintenance of Chromosomes (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion. SMCs modulate DNA supercoiling, but it has remained incompletely understood how this is achieved. Here we present a single-molecule magnetic tweezers assay that directly measures how much twist is induced by an individual SMC in each loop-extrusion step. We demonstrate that all three SMC complexes induce the same large negative twist (i.e., a linking number change {Delta}Lk of -0.6 at each loop-extrusion step) into the extruded loop, independent of step size. Using ATP-hydrolysis mutants and non-hydrolysable ATP analogues, we find that ATP binding is the twist-inducing event during the ATPase cycle, which coincides with the force-generating loop-extrusion step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-loop-extrusion mechanism among these SMC complexes.

biophysics↗

bioRxiv2024

Cohesin supercoils DNA during loop extrusion

Cohesin extrudes genomic DNA into loops that promote chromatin assembly, gene regulation and recombination. Here we show that cohesin introduces negative supercoils into extruded DNA. Supercoiling requires engagement of cohesins ATPase heads, DNA clamping by these heads, and a DNA binding site on cohesins hinge, indicating that cohesin supercoils DNA when constraining it between the hinge and the clamp. Our results suggest that DNA extrusion stops once cohesin reaches its stall torque during supercoiling, and a cohesin mutant predicted to stall at lower torque forms shorter loops in cells. These results indicate that supercoiling is an integral part of the loop extrusion mechanism and that cohesin controls genome architecture not only by looping DNA but also by supercoiling it.

biochemistry↗

bioRxiv2023

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

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.

biophysics↗

bioRxiv2023

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

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.

biophysics↗

bioRxiv2022

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

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 cohesins 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, where 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.

biophysics↗

bioRxiv2021

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

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 the early biofilm structural framework. Together, our findings and investigative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.

microbiology↗

bioRxiv2020

Resolving the step size in condensin-driven DNA loop extrusion identifies ATP binding as the step-generating process

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.

biophysics↗

bioRxiv2020

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

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA complex and the dimensions of the RdRp nucleic-acid-binding channel. In cells, recombination was stimulated by the presence of a pyrazine-carboxamide ribonucleotide. The effect of the drug on recombination was diminished for a recombination-defective virus, but this virus was not resistant to the drug. The discovery that a ribonucleotide analogue can induce copy-back RNA synthesis suggests that this third mechanistic class of compounds may function by promoting formation of defective viral genomes. This study identifies RdRp-catalyzed intra- and intermolecular template switching as a viable new mechanistic target with potentially broad-spectrum appeal.

microbiology↗

bioRxiv2019

A unifying mechanistic model of bacterial transcription with three interconnected pause states and non-diffusive backtrack recovery

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 understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examined the E. coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identified, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We further identified two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlie elongation-rate switching, and RNA cleavage deficiency that underpins different long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.

biophysics↗

bioRxiv2018

Predicting Intra- and Intertypic Recombination in Enterovirus 71

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: (1) EV-A71 strain-type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (2) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase (RdRp); (3) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination suggesting conservation in mechanism(s); and (4) sequencing of intertypic recombinant genomes indicates that template-switching is by a mechanism that requires 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.

microbiology↗