Biology subjects

Pastrana, C. L.

Publications and source records attributed to Pastrana, C. L..

7 recordsLinked to original sources

bioRxiv2026

Understanding the role of chromatin accessibility and compaction on loop extrusion dynamics

Loop extrusion by SMC complexes is a fundamental mechanism regulating the three-dimensional genome organization, but how these molecular motors navigate through the crowded, nucleosome-covered chromatin fiber remains an open question. Here, we develop a stochastic biophysical model of loop extrusion that explicitly couples motor stepping dynamics to chromatin accessibility and compaction. First, we systematically analyze the role of accessibility on its dynamics across homogeneous, periodic, and random roadblock positioning along chromatin. This comprehensive analysis reveals distinct dynamic regimes: phase-dependent kinetics in periodic arrays where motor step size aligns with obstacle spacing, and anomalous sub-linear progression in disordered environments due to rare but long-lived trapping events. Then, we contextualize our framework to nucleosome-like arrays. Our simulations reveal a non-intuitive dual role for nucleosomes: while they hinder extruder progression by blocking access to linker DNA, their high compaction packs more base pairs per physical distance, enabling larger genomic displacements per step. Strikingly, the resulting base-pair extrusion speed displays a non-monotonic dependence on nucleosome spacing, reaching a maximum at intermediate linker lengths (10-70 bp) that can exceed the speed on naked DNA. Finally, in support of this mechanism, analysis of yeast genomic data shows that a higher nucleosome occupancy between cohesin-associated regions correlates with stronger contact enrichment, consistent with compaction-enhanced extrusion efficiency in vivo. These findings establish chromatin architecture as a tunable regulator of loop extrusion, explaining the comparable speeds observed in vitro and in vivo and providing a quantitative framework for understanding how genome organization emerges from the interplay between motor kinetics and chromatin structure.

biophysics↗

bioRxiv2025

Force Generation by Enhanced Diffusion in Enzyme-Loaded Vesicles

Recent experiments have shown that the diffusion coefficient of some metabolic enzymes increases with the concentration of their cognate substrate, a phenomenon known as enhanced diffusion. In the presence of substrate gradients, enhanced diffusion induces enzymatic drift, resulting in a non-homogeneous enzyme distribution. Here, we study the effects of enhanced diffusion on enzyme-loaded vesicles placed in external substrate gradients using a combination of computer simulations and analytical modeling. We observe that the spatially inhomogeneous enzyme profiles generated by enhanced diffusion result in a pressure gradient across the vesicle, which leads to macroscopically observable effects, such as deformation and self-propulsion of the vesicle. Our analytical model allows us to characterize dependence of the velocity of propulsion on experimentally tunable parameters. The effects predicted by our work provide an avenue for further validation of enhanced diffusion, and might be leveraged for the design of novel synthetic cargo transporters, such as targeted drug delivery systems.

biophysics↗

bioRxiv2021

Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

Faithful segregation of bacterial chromosomes relies on the ParABS partitioning system and the SMC complex. In this work, we used single molecule techniques to investigate the role of cytidine triphosphate (CTP) binding and hydrolysis in the critical interaction between centromere-like parS DNA sequences and the ParB CTPase. Using a combined dual optical tweezers confocal microscope, we observe the specific interaction of ParB with parS directly. Binding around parS is enhanced 4-fold by the presence of CTP or the non-hydrolysable analogue CTP{gamma}S. However, ParB proteins are also detected at a lower density in distal non-specific regions of DNA. This requires the presence of a parS loading site and is prevented by roadblocks on DNA, consistent with one dimensional diffusion by a sliding clamp. Magnetic tweezers experiments show that the spreading activity, which has an absolute requirement for CTP binding but not hydrolysis, results in the condensation of parS-containing DNA molecules at low nanomolar protein concentrations. We propose a model in which ParB-CTP-Mg2+ complexes move along DNA following loading at parS sites and protein:protein interactions result in the localised condensation of DNA within ParB networks.

biophysics↗

bioRxiv2019

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, their response to forces remains unknown and the variability of their flexibility reported for different length scales has precluded a comprehensive description of the mechanical properties of these molecules. Here, we rationalize the mechanical properties of A-tracts across multiple length scales using a combination of single-molecule experiments and theoretical polymer models applied to DNA sequences present in the C. elegans genome. Atomic Force Microscopy imaging shows that phased A-tracts induce long-range ([~]200 nm) bending. Moreover, the enhanced bending originates from an intrinsically bent structure rather than as a consequence of larger flexibility. In support of this, our data were well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments confirm that the observed bent is intrinsic to the sequence and does not rely on particular ionic conditions. Using optical tweezers, we assess the local rigidity of A-tracts at high forces and unravel an unusually stiff character of these sequences, as quantified by their large stretch modulus. Our work rationalizes the complex multiscale flexibility of A-tracts, shedding light on the cryptic character of these sequences.

biophysics↗

bioRxiv2019

Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA 1. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with re-opening of the gates to release DNA and to recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

microbiology↗

bioRxiv2018

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. In our previous work (Fisher et al., 2017), we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks. However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualize ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerization with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks.\n\nSignificance StatementUsing combined Magnetic Tweezers and TIRF microscopy we show that the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA.

molecular biology↗

bioRxiv2017

The C-Terminal Domain Of ParB Is Critical For Dynamic DNA Binding And Bridging Interactions Which Condense The Bacterial Centromere

The ParB protein forms DNA bridging interactions around parS to form networks which condense DNA and earmark the bacterial chromosome for segregation. The mechanism underlying the formation of ParB nucleoprotein complexes is unclear. We show here that the central DNA binding domain is essential for anchoring at parS, and that this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks.

biochemistry↗