Biology subjects

Roca, J.

Publications and source records attributed to Roca, J..

5 recordsLinked to original sources

bioRxiv2026

DNA catenation is essential for Sister Chromatid Cohesion

From S phase until anaphase, sister chromatids remain physically linked to ensure accurate chromosome segregation. This linkage, called sister chromatid cohesion, must withstand the pulling forces generated by the mitotic spindle and is thought to require entrapment of sister DNAs inside the ring-shaped cohesin complex. DNA catenations that arise naturally during replication could also tether sister chromatids, but whether they contribute to cohesion has remained unresolved. Here, using budding yeast, we show that cohesin-mediated sister DNA entrapment alone cannot withstand spindle-generated pulling forces and that robust sister chromatid cohesion requires DNA catenation. Selective removal of DNA catenations causes catastrophic cohesion loss, delays chromosome biorientation and increases chromosome mis-segregation, even when cohesin rings remain intact. Importantly, DNA catenation similarly underpins force-resistant cohesion in metazoan chromosomes. Our findings fundamentally redefine the physical basis of sister chromatid cohesion by establishing DNA catenation as an evolutionarily conserved component of the force-resistant linkage between sister chromatids that enables accurate chromosome segregation.

molecular biology↗

bioRxiv2023

Nucleosomal DNA has topological memory

One fundamental yet elusive aspect of the chromosome architecture is the constrained topolome, which refers to how chromatin elements restrain DNA topology. Nucleosomes stabilise negative DNA supercoils, with most nucleosomes typically restraining a DNA linking number difference ({Delta}Lk) of about -1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculated the {Delta}Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we placed each nucleosome in a circular minichromosome and performed Topo-seq, a novel high-throughput procedure to inspect the topology of circular DNA libraries in a single gel electrophoresis. We found that nucleosomes inherently restrain distinct {Delta}Lk values depending on their genomic origin. Nucleosome DNA topologies differ significantly at gene bodies ({Delta}Lk=-1.29), intergenic regions ({Delta}Lk=-1.23), rDNA genes ({Delta}Lk=-1.24) and telomeric regions ({Delta}Lk=-1.07). Nucleosomes nearby the transcription start and termination sites also exhibit singular DNA topologies. These findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists even when the nucleosome is relocated. This imprinting contributes to nucleosome functional roles and chromatin folding architectures.

molecular biology↗

bioRxiv2022

Condensin pinches a short negatively supercoiledDNA loop during each round of ATP usage

Condensin extrudes DNA loops using an ATP-dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain DNA positive supercoils. However, in experimental conditions that sustain DNA loop extrusion, condensin restrains negative supercoils. Namely, upon ATP-mediated loading onto DNA, each condensin constrains a DNA linking number difference ({Delta}Lk) of -0.4. This {Delta}Lk increases to -0.8 during ATP binding and resets to -0.4 upon ATP hydrolysis. These {Delta}Lk values reflect the transient formation of a short left-handed loop of DNA, which is not the extruding loop. We conclude that, upon condensin ATPase-head engagement, a segment of DNA is pinched to form a short negatively supercoiled loop, which can be subsequently merged with the extruding loop. Such "pinch and merge" mechanism implies that the DNA is transferred between two dynamic DNA-binding sites while anchored at a third site.

molecular biology↗

bioRxiv2022

Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfqs integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry (nMS) coupled with surface-induced dissociation (SID) and molecular dynamics (MD) simulations to disentangle the arrangement of the CTDs and their impact on the stability of E. coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding. Significance StatementHfq is a protein hexamer necessary for gene regulation by non-coding RNA in bacteria, during infection or under stress. In the cell, Hfq must distinguish its RNA partners from many similar nucleic acids. Mass spectrometry dissociation patterns, together with molecular dynamics simulations, showed that flexible extensions of each Hfq subunit form a dense network that interconnects the entire hexamer. This network is disrupted by RNA binding, but the lost interactions are compensated by RNAs that contact multiple Hfq subunits. By measuring interactions that are too irregular to be counted by other methods, mass spectrometry shows how flexible protein extensions help chaperones like Hfq recognize their RNA partners in the messy interior of the cell.

biophysics↗

bioRxiv2017

Monovalent ions modulate the flux through multiple folding pathways of an RNA pseudoknot

The functions of RNA pseudoknots (PKs), which are minimal tertiary structural motifs and an integral part of several ribozymes and ribonucleoprotein complexes, are determined by their structure, stability and dynamics. Therefore, it is important to elucidate the general principles governing their thermodynamics/folding mechanisms. Here, we combine experiments and simulations to examine the folding/unfolding pathways of the VPK pseudoknot, a variant of the Mouse Mammary Tumor Virus (MMTV) PK involved in ribosomal frameshifting. Fluorescent nucleotide analogs (2-aminopurine and pyrrolocytidine) placed at different stem/loop positions in the PK, and laser temperature-jump approaches serve as local probes allowing us to monitor the order of assembly of VPK with two helices with different intrinsic stabilities. The experiments and molecular simulations show that at 50 mM KCl the dominant folding pathway populates only the more stable partially folded hairpin. As the salt concentration is increased a parallel folding pathway emerges, involving the less stable hairpin structure as an alternate intermediate. Notably, the flux between the pathways is modulated by the ionic strength. The findings support the principle that the order of PK structure formation is determined by the relative stabilities of the hairpins, which can be altered by sequence variations or salt concentrations. Our study not only unambiguously demonstrates that PK folds by parallel pathways, but also establishes that quantitative description of RNA self-assembly requires a synergistic combination of experiments and simulations.\n\nSIGNIFICANCEThe assembly mechanism of RNA, vital to describing their functions, depends on both the sequence and the metal ion concentration. How the latter influences the folding trajectories remains an important unsolved problem. Here, we examine the folding pathways of an RNA pseudoknot (PK) with key functional roles in transcription and translation, using a combination of experiments and simulations. We demonstrate that the PK, consisting of two helices with differing stabilities, folds by parallel pathways. Surprisingly, the flux between them is modulated by monovalent salt concentration. Our work shows that the order of assembly of PKs is determined by the relative stability of the helices, implying that the folding landscape can be controlled by sequence and ion concentration.

biophysics↗