Biology subjects

Pobegalov, G.

Publications and source records attributed to Pobegalov, G..

7 recordsLinked to original sources

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

A new material constant of DNA is the major determinant of looping dynamics

DNA bending and looping is crucial for gene expression, packaging, and chromatin organisation, as well as the design of artificial nanomaterials and devices. But what determines how quickly DNA bends? While DNAs static flexibility is well-characterised by its persistence length, we lack an understanding of how quickly DNA responds to mechanical forces: remarkably current semiflexible polymer theory based on solvent dissipation underestimates spontaneous looping times by ~1000-fold. By analysing fluctuations of DNA several kilobases long and developing new theory for bending dissipation in semiflexible polymers, we show DNA bending dynamics cannot be explained by solvent friction alone and requires significant contributions from intramolecular friction. The theory defines a new material constant of DNA -- the bending friction, which we determine to be{zeta} B = 241 {+/-} 17 g nm3/ms. Strikingly, our measurement does not depend on the buffer ionic conditions. We predict bending friction will dominate DNA dynamics between {approx} 50 nm and 420 nm and significantly longer under external force. We show that mean first passage time calculations are greatly simplified when bending friction dominates and so using this constant, with no fitting parameters, we accurately predict the slow experimental spontaneous looping times. Our discovery of significant bending dissipation is unexpected as DNA has no obvious large (> kBT) internal energy barriers. The salt-independence of this dissipation also rules out long range electrostatic interactions as its origins. Instead our findings point to a complex local energy landscape for bending and a potential previously unappreciated role of water binding DNA constraining its local mobility. Our findings radically change our understanding of DNA dynamics and reveal DNA as a viscoelastic semiflexible polymer with dramatically slower dynamics compared to an ideal elastic rod. This work establishes bending friction as a fundamental material property that must underpin any model of DNA dynamics in biology, physics, and nanotechnology.

biophysics↗

bioRxiv2024

Force-transducing molecular ensembles at growing microtubule tips control mitotic spindle size

Mitotic spindle is a complex bipolar cellular structure that ensures chromosomes segregation between dividing cells. Correct spindle size is required for the accurate segregation and successful passing of genomes to the newly formed cells. The spindle size is believed to be controlled by mechanical forces generated by molecular motors and non-motor proteins acting in the spindle microtubule overlaps. However, how forces generated by individual proteins enable bipolar spindle organization is not well understood. Here, we developed tools to measure contributions of individual molecules to this force balance. We show that microtubule tip-trackers act synergetically at microtubule tips with minus-end directed motors to produce a system that can generate both pushing and pulling forces. We show that this system harnesses forces generated by growing tips of spindle microtubules and provides unique contribution to the force balance distinct from other force generators because it acts at microtubule tips rather than in microtubule overlaps. We show that this system alone can establish stable bipolar organization in vitro and in mitotic spindles in human cells. Our results pave the way for understanding how mechanical forces in spindles can be fine-tuned to control the fidelity of chromosome segregation.

biophysics↗

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

Single-molecule studies reveal the off-pathway elemental pause state as a target of streptolydigin inhibition of RNA polymerase and its dramatic enhancement by Gre factors

Antibiotic streptolydigin (Stl) inhibits bacterial transcription by blocking the trigger loop folding in the active center of RNA polymerase (RNAP), which is essential for catalysis. We use acoustic force spectroscopy to characterize the dynamics of transcription elongation in ternary elongation complexes of RNAP (ECs) in the presence of Stl at a single-molecule level. We found that Stl induces long-lived stochastic pauses while the instantaneous velocity of transcription between the pauses is unaffected. Stl enhances the short-lived pauses associated with an off-pathway elemental paused state of the RNAP nucleotide addition cycle. Unexpectedly, we found that transcript cleavage factors GreA and GreB, which were thought to be Stl competitors, do not alleviate the streptolydigin-induced pausing; instead, they synergistically increase transcription inhibition by Stl. This is the first known instance of a transcriptional factor enhancing antibiotic activity. We propose a structural model of the EC-Gre-Stl complex that explains the observed Stl activities and provides insight into possible cooperative action of secondary channel factors and other antibiotics binding at the Stl-pocket. These results offer a new strategy for high-throughput screening for prospective antibacterial agents.

biophysics↗

bioRxiv2022

A new insight into RecA filament regulation by RecX from the analysis of conformation-specific interactions

RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on single-stranded DNA (ssDNA) in an ATP dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of (E. coli) RecA-ssDNA filaments by RecX (E. coli) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

molecular biology↗

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↗