Biology subjects

Uhlmann, F.

Publications and source records attributed to Uhlmann, F..

14 recordsLinked to original sources

bioRxiv2026

DNA entry into the cohesin ring through the kleisin N-gate

The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion, a prerequisite for faithful chromosome segregation during cell divisions. How DNA enters the cohesin ring remains incompletely understood. Both the interface between Psm3Smc3 and the Rad21Scc1 N-terminus - the N-gate - and the SMC hinge interface - the hinge gate - can open in vitro to entrap DNA. Here, we combine biochemical with in vivo functional assays in the fission yeast S. pombe to probe the DNA entry mechanism. Locking the hinge gate does not interfere with cohesin loader-and ATP hydrolysis-dependent DNA entrapment. Conversely, cohesin with a tightly locked N-gate entraps DNA in vitro but does not load onto chromatin or establish sister chromatid cohesion in vivo. The cohesin ATPase is functionally coupled to N-gate, but not hinge-gate, opening. Our results put forward the N-gate as the primary DNA entry gate into the cohesin ring.

molecular biology↗

bioRxiv2026

Symmetry and force response of cohesin loop extrusion are determined by diffusion of its motor and anchor domains

Cohesin, a Structural Maintenance of Chromosomes (SMC) complex is thought to organize genomes by generating DNA loops, yet the molecular basis of loop extrusion remains incompletely understood. Here, we show that a single S. pombe cohesin complex extrudes DNA loops both symmetrically or asymmetrically, depending on the external force applied to DNA and that the mode and speed are controlled by stochastic switching of the cohesin motor between driving and diffusive states. We directly measured the weak forces generated during cohesin-mediated loop extrusion while simultaneously visualizing DNA loops and found that forces as low as 0.05 pN pull DNA out of cohesin loop without disrupting cohesin-DNA interaction. We further identified the Scc3 subunit as a diffusive DNA anchor required for loop extrusion and showed that its physical separation from the motor domain by unstructured regions of Scc1 promotes loop-extrusion initiation. Together, our results support a model in which cohesin loop extrusion is limited and tuned by diffusive motion of both anchor and motor modules, providing a mechanistic framework for how weak and diffusive cohesin-DNA interactions could modulate cohesin function in chromatin organization.

biophysics↗

bioRxiv2025

Regulation of cell proliferation by a novel feedback system on Cdk function

The proliferation of eukaryotic cells is regulated by a complex network of regulatory systems that promotes efficient cell cycle progression and ensures proper responses to the environment. Despite this complexity, the core inputs that are necessary and sufficient for robust alternation of DNA replication and mitosis are surprisingly simpler than anticipated. Indeed, fission yeast cells operating with an engineered minimal cell cycle network that lacks the highly conserved Wee1+Cdc25 feedback loops on Cdk1 function are viable, although slow growing. This provides a unique entry for evaluating how such simplified cells can evolve and improve their proliferation potential while exploring unknown mechanisms modulating cell cycle progression. Taking advantage of this model, we applied laboratory evolution assays to minimal fission yeast backgrounds and selected for the emergence of faster growing populations. We found that loss of the small disordered protein Spo12 brings about enhanced population growth in cells lacking the Wee1+Cdc25 mitotic switch. Importantly, we demonstrate that Spo12 defines a new and conserved family of inhibitors of the Cdk-counteracting phosphatase PP2A that are directly regulated by Cdk-dependent phosphorylation. Our results also reveal a trade-off associated with Spo12-dependent regulation, which may have implications for our understanding of the principles underlying the evolution of cell cycle control. Finally, our study highlights how combining simplified circuits with experimental evolution allows for uncovering regulatory elements that may be obscured by network complexity.

cell biology↗

bioRxiv2025

Investigations into fission yeast chromosome size determinants

Mitotic chromosome dimensions differ between organisms, and they differ within a species between developmental stages. The physiological determinants of chromosome size remain poorly understood. Here, we investigate chromosome size determinants in the fission yeast Schizosaccharomyces pombe. Super-resolution microscopy and semi-automated measurements reveal that cell or nuclear volume in interphase, or the time spent in mitosis (both previously proposed chromosome size determinants), have little influence on resultant chromosome dimensions. Instead, levels of the chromosomal condensin complex affect chromosome size, with increasing condensin levels resulting in more compact, shorter and thinner, chromosomes. These observations inform our understanding of how chromosome dimensions are controlled in an organism. They suggest that a chromosome-intrinsic mechanism sets chromosome size, more so than the environment in which chromosomes find themselves in.

cell biology↗

bioRxiv2025

Biochemical reconstitution of sister chromatid cohesion establishment during DNA replication

Concomitant with DNA replication, the ring-shaped cohesin complex encircles both newly synthesized sister chromatids, enabling their faithful segregation during cell divisions. Our molecular understanding of how cohesin co-entraps both replication products remains incomplete. Here, we reconstitute sister chromatid cohesion establishment using purified budding yeast proteins. Cohesin rings, initially loaded onto template DNA, remain DNA-bound during DNA synthesis and encircle both replication products. Additionally, DNA replication instigates new cohesin recruitment, as a second pathway that generates sister chromatid cohesion. In both scenarios, cohesin often embraces only one of the two replication products, suggestive of a two-step sister chromatid capture mechanism. Sister chromatid co-entrapment occurs independently of replication fork-associated cohesion establishment factors, suggesting a role for the latter during chromatin replication or in facilitating the subsequent cohesin acetylation reaction. Our results make sister chromatid cohesion establishment amenable to direct experimental exploration.

molecular biology↗

bioRxiv2024

Progressive chromosome shape changes during cell divisions, recapitulated by loop capture simulations

Mitotic chromosomes give genome portions the required compaction and mechanical stability for faithful inheritance during cell divisions. Here, we record human chromosome dimensions from their appearance in prophase over successive times in a mitotic arrest. Chromosomes first appear long and uniformly thin. Then, individual chromosome arms become discernible, which continuously shorten and thicken - the longer a chromosome arm, the thicker it becomes. The observed chromosome arm length to width relationship can be described by a power law with progressively increasing exponent. In the search for a molecular explanation of this behavior, the popular loop extrusion model provides no obvious means by which longer arms become thicker. Instead, we find that simulations of an alternative loop capture model recapitulate key features of our observations, including the gradually developing arm length to width relationship. Our analyses portray chromosomes as out-of-equilibrium structures in the process of transitioning towards, but on biologically relevant time scales not typically reaching, steady state.

biophysics↗

bioRxiv2024

Replisome passage through the cohesin ring

Following eukaryotic genome replication, the two newly synthesised sister chromatids remain paired by the ring-shaped cohesin complex, enabling their faithful segregation to daughter cells during cell divisions1. Cohesin topologically embraces DNA already before DNA replication2-5, and replisome passage through the cohesin ring is thought of as a fail-safe mechanism ensuring that cohesin entraps both replication products6,7. Whether replisomes indeed pass through cohesin rings remains unknown. Here, we use biochemical reconstitution4,8,9 and single molecule fluorescence microscopy to directly visualise replisome-cohesin encounters. We find that the translocating eukaryotic replicative Cdc45-Mcm2-7-GINS (CMG) helicase is frequently an obstacle to cohesin, but that the likelihood with which CMG passes cohesin increases in the presence of replisome components with known sister chromatid cohesion functions7,10-14, or by preventing cohesin from freely sliding along the template DNA. Cohesin retains topological DNA entrapment during CMG passage, suggesting that the helicase passes through the ring. The passage frequency increases further when fully reconstituted replisomes encounter cohesin rings, resulting in successful establishment of cohesion between the two replication products. Our findings demonstrate the existence of a simple mechanism that links genome replication with chromosome segregation, replisome passage through cohesin rings.

molecular biology↗

bioRxiv2023

Cohesin chromatin loop formation by an extrinsic motor

The ring-shaped cohesin complex topologically entraps two DNAs to establish sister chromatid cohesion1-3. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation4-7, which cohesin is thought to achieve by actively extruding DNA loops without topologically entrapping DNA8-11. The loop extrusion hypothesis finds motivation from in vitro observations12-14 - whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, as well as acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion model and point to an alternative mechanism for cohesin-dependent chromatin organisation. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesins two roles in chromosome segregation and interphase genome organisation.

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

Telomere-to-telomere Schizosaccharomyces japonicus genome assembly reveals hitherto unknown genome features

Schizosaccharomyces japonicus belongs to the single-genus class Schizosaccharomycetes, otherwise known as fission yeasts. As part of a composite model system with its widely studied S. pombe sister species, S. japonicus has provided critical insights into the workings and the evolution of cell biological mechanisms. Furthermore, its divergent biology makes S. japonicus a valuable model organism in its own right. However, the currently available short-read genome assembly contains gaps and has been unable to resolve centromeres and other repeat-rich chromosomal regions. Here we present a telomere-to-telomere long-read genome assembly of the S japonicus genome. This includes the three megabase-length chromosomes, with centromeres hundreds of kilobases long, rich in 5S ribosomal RNAs, transfer RNAs, long terminal repeats, and short repeats. We identify a gene-sparse region on chromosome 2 that resembles a 331 kb centromeric duplication. We revise the genome size of S. japonicus to at least 16.6 Mb and possibly up to 18.12 Mb, at least 30% larger than previous estimates. Our whole genome assembly will support the growing S. japonicus research community and facilitate research in new directions, including centromere and DNA repeat evolution, and yeast comparative genomics. Take-awayO_LIA telomere-to-telomere genome assembly of the fission yeast S. japonicus C_LIO_LIChromosome 2 harbours a previously unknown second centromere-like region C_LIO_LIThe estimated genome size of S. japonicus may be up to 18.12 Mb C_LI

genomics↗

bioRxiv2021

A Brownian ratchet model for DNA loop extrusion by the cohesin complex

The cohesin complex topologically encircles DNA to promote sister chromatid cohesion. Alternatively cohesin extrudes DNA loops, thought to reflect chromatin domain formation. Here, we propose a structure-based model explaining both activities, supported by biochemical experiments. ATP and DNA binding to cohesin promote conformational changes that guide DNA through a kleisin gate into a DNA gripping state. Two HEAT-repeat DNA binding modules, associated with cohesins heads and hinge, are now juxtaposed. ATP hydrolysis disassembles the gripping state, allowing unidirectional hinge module movement to complete topological DNA entry. Without initial kleisin gate passage, biased hinge module motion during gripping state resolution creates a Brownian ratchet that drives loop extrusion. Molecular-mechanical simulations of gripping state formation and resolution cycles recapitulate experimentally observed DNA loop extrusion characteristics. Our model extends to asymmetric and symmetric loop extrusion, as well as z-loop formation. Loop extrusion by biased Brownian fluctuations has important implications for chromosomal cohesin function.

biophysics↗

bioRxiv2020

Computational and experimental analyses of mitotic chromosome formation pathways in fission yeast

Underlying higher order chromatin organization are Structural Maintenance of Chromosomes (SMC) complexes, large protein rings that entrap DNA. The molecular mechanism by which SMC complexes organize chromatin is as yet incompletely understood. Two prominent models posit that SMC complexes actively extrude DNA loops (loop extrusion), or that they sequentially entrap two DNAs that come into proximity by Brownian motion (diffusion capture). To explore the implications of these two mechanisms, we perform biophysical simulations of a 3.76 Mb-long chromatin chain, the size of the long S. pombe chromosome I left arm. On it, the SMC complex condensin is modeled to perform loop extrusion or diffusion capture. We then compare computational to experimental observations of mitotic chromosome formation. Both loop extrusion and diffusion capture can result in native-like contact probability distributions. In addition, the diffusion capture model more readily recapitulates mitotic chromosome axis shortening and chromatin density enrichment. Diffusion capture can also explain why mitotic chromatin shows reduced, as well as more anisotropic, movements, features that lack support from loop extrusion. The condensin distribution within mitotic chromosomes, visualized by stochastic optical reconstruction microscopy (STORM), shows clustering predicted from diffusion capture. Our results inform the evaluation of current models of mitotic chromosome formation.

biophysics↗

bioRxiv2020

Phase separation induced by cohesin SMC protein complexes

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATP-independent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summaryYeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.

biophysics↗

bioRxiv2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

molecular biology↗