Biology subjects

van der Torre, J.

Publications and source records attributed to van der Torre, J..

12 recordsLinked to original sources

bioRxiv2026

Intercalator effects on DNA loop extrusion by SMC proteins

Intercalating dyes are widely used to visualize DNA in single-molecule experiments, but their effects on DNA-processing proteins remain incompletely characterized. Here, we study the effects of intercalators on DNA loop extrusion by SMC complexes. Intercalating dyes become fluorescent upon DNA binding, enabling direct visualization of extruded DNA loops. As dye intercalation alters the biophysical properties of DNA by lengthening and unwinding it, we examine how that modifies loop extrusion. We systematically characterize the effects of intercalators, and find that they stiffen DNA, partly neutralize its charge, and can substantially reduce the binding and ATPase activity of SMCs. We assess four dyes and conclude that SYBR Gold and SYTOX Orange are less invasive intercalators than SYTOX Green or YOYO-1. While loop extrusion is qualitatively not affected by intercalation, quantitative values for SMC binding and looping rates differ significantly from those in the absence of intercalators. We present a new dye-free loop-extrusion assay (utilizing local fluorescent spots along DNA), and find that loop extrusion proceeds at 1.75{+/-}0.26 kb/s for S. cerevisiae condensin without dyes, while adding intercalators leads to a two-fold lower rate. Our findings establish that intercalators quantitatively modify loop extrusion parameters, and we offer practical recommendations for minimizing their impact.

biophysics↗

bioRxiv2026

A modular DNA toolkit for single-molecule biophysics

Long, customizable DNA molecules are an essential tool for in vitro single-molecule biophysics. Most studies still rely on the {lambda}-phage genome, which has a fixed sequence, a non-uniform GC distribution, and limited options for modification. To overcome these limitations, we developed a modular DNA (ModDNA) toolkit that integrates in silico sequence design with cloning, to create long (up to 100 kb) DNA molecules with entirely user-defined sequences. We used a computational workflow with curated databases to design a motif-depleted sequence backbone, into which any desired sequence can be inserted at any position. Golden Gate assembly then builds these sequences into constructs, which we extend iteratively in defined increments up to 100 kb. We show precise placement of local features, e.g. a fluorophore, a bound protein, and a DNA-origami structure, and illustrate the construct's utility in single-molecule visualization assays, including DNA loop extrusion by human condensin I. ModDNA offers a scalable, sequence-programmable alternative to {lambda}-DNA and other restrictive substrates. More broadly, the motif-aware feature-free design connects single-molecule biophysics to emerging efforts in machine-guided regulatory-sequence design and synthetic-genome engineering, where controlled programmable sequence context is increasingly essential.

biophysics↗

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↗

bioRxiv2023

SMC motor proteins extrude DNA asymmetrically and contain a direction switch

Structural Maintenance of Chromosomes (SMC) complexes organize the genome via DNA loop extrusion. While some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, while the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. STAG1 stabilizes NIPBL on cohesin, preventing NIPBL turnover and direction switches. The findings reveal that SMCs, surprisingly, contain a direction switch subunit. HighlightsO_LIAll eukaryotic SMC complexes extrude DNA asymmetrically. C_LIO_LIApparent symmetric loop extrusion is the result of frequent direction switches. C_LIO_LIn human cohesin, loop-extrusion direction changes require exchange of NIPBL. C_LIO_LISTAG1 stabilizes NIPBL on human cohesin. C_LI

molecular biology↗

bioRxiv2023

Telomere protein arrays stall DNA loop extrusion by condensin

DNA loop extrusion by SMC proteins is a key process underlying chromosomal organization. It is unknown how loop extruders interact with telomeres where chromosome ends are covered with a dense array of tens of neighboring DNA-binding proteins. Using complementary in vivo and in vitro single-molecule approaches, we study the interaction between loop-extruding condensin and arrays of Rap1, the double-stranded-DNA-binding telomeric protein of Saccharomyces cerevisiae. We show that dense linear Rap1 arrays can completely halt DNA loop extrusion, where the blocking efficiency depends on the array length and the DNA gap size between neighboring proteins. In cells, Rap1 arrays in the chromosome are found to act as contact insulators and to accumulate condensin at their borders, with direct implications for the resolution of dicentric chromosomes produced by telomere fusions. Our findings show that linear arrays of DNA-bound proteins can efficiently halt DNA loop extrusion by SMC proteins, which may impact a wide range of cellular processes from telomere functions to transcription and DNA repair.

molecular biology↗

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↗

bioRxiv2022

Extracting and characterizing protein-free megabasepair DNA for in vitro experiments

Chromosome structure and function is studied in cells using imaging and chromosome-conformation-based methods as well as in vitro with a range of single-molecule techniques. Here we present a method to obtain genome-size (megabasepair length) deproteinated DNA for in vitro studies, which provides DNA substrates that are two orders of magnitude longer than typically studied in single-molecule experiments. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97-100% of DNA-binding proteins are removed from the sample. Upon protein removal, we observed an increase in the radius of gyration of the DNA polymers, while quantification of the fluorescence intensities showed that the length of the DNA objects remained megabasepair sized. In first proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labelled DNA locus. These results indicate the practical feasibility of a genome-in-a-box approach to study chromosome organization from the bottom up.

biophysics↗

bioRxiv2021

ParS-independent recruitment of the bacterial chromosome-partitioning protein ParB

The ParABS system plays an essential role in prokaryotic chromosome segregation. After loading at the parS site on the genome, ParB proteins rapidly redistribute to distances of ~15 kb away from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA that is loaded with hundreds of proteins. Using single-molecule in vitro visualization, we here show that, unexpectedly, ParB can load onto DNA independently and distantly of parS, whereby loaded ParB molecules are themselves able to recruit additional ParB proteins from bulk. Strikingly, this recruitment can occur in-cis but also in-trans whereby, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by Molecular Dynamics simulations which also show that cooperative ParB-ParB recruitment enhances spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation which is vital for the bacterial cell cycle.

biophysics↗

bioRxiv2021

SMC complexes can traverse physical roadblocks bigger than their ring size

The ring-shaped structural-maintenance-of-chromosomes (SMC) complexes condensin and cohesin extrude loops of DNA as a key motif in chromosome organization. It remains, how ever, unclear how these SMC motor proteins can extrude DNA loops in chromatin that is bound with proteins. Here, using in vitro single-molecule visualization, we show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to DNA loop extrusion by yeast condensin. Strikingly, we find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, can be translocated into DNA loops during condensin-driven extrusion. Similarly, human cohesin can pass 200 nm particles during loop extrusion, which even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open up to entrap DNA. These findings disqualify all common loop-extrusion models where DNA passes through the SMC rings (pseudo)topologically, and instead point to a nontopological mechanism for DNA loop extrusion. One-sentence summaryHuge DNA-bound roadblocks can be incorporated into SMC-extruded DNA loops, pointing to a nontopological mechanism for loop extrusion.

biophysics↗

bioRxiv2021

Condensin-driven loop extrusion on supercoiled DNA

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, e.g., the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping is stimulated by positive supercoiled DNA where it preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects all nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.

biophysics↗