bioRxiv Science⌕ Search

Biology subjects

Aragon, L.

Publications and source records attributed to Aragon, L..

12 recordsLinked to original sources
bioRxiv2026

Human FACT complex coordinates cohesin and transcription on chromatin

The spatial organisation of eukaryotic genomes is critical for coordinating DNA-dependent processes such as transcription and replication. Cohesin contributes to this organisation by extruding chromatin loops; however, how cohesin achieves processive translocation along nucleosome-dense chromatin in vivo remains poorly understood. Here, we identify the histone chaperone FACT as a regulator of cohesin dynamics in human cells. We show that the FACT subunit SUPT16H colocalises and interacts with cohesin on chromatin. Depletion of FACT impairs cohesin translocation from its loading sites, leading to reduced chromatin looping and decreased boundary strength of topologically associating domains (TADs), as revealed by Hi-C. In addition, FACT depletion results in the accumulation of cohesin within gene bodies that are not engaged in loop formation, which is associated with reduced transcription. Importantly, degradation of cohesin in FACT-depleted cells restores transcriptional output, indicating that cohesin mislocalisation underlies this effect. Together, these findings demonstrate that FACT facilitates cohesin translocation along chromatin, promoting efficient loop extrusion while preventing its aberrant accumulation within transcribed regions. This work provides mechanistic insight into how chromatin structure modulates cohesin function to coordinate genome organisation with transcription in mammalian cells.

biochemistry↗
bioRxiv2026

Negative supercoiling facilitates human TOP3α-mediated decatenation of DNA braids via catenation junction melting

DNA catenation between sister chromatids arises during replication and must be efficiently resolved to ensure faithful chromosome segregation. The removal of DNA catenation is generally attributed to the type II topoisomerase TOP2, which catalyses double-stranded DNA passage reactions and can therefore directly remove inter-DNA linkages. By contrast, the type IA topoisomerase TOP3 catalyses strand passage through transient breaks in single-stranded DNA. Here, using single-molecule optical tweezers and fluorescence microscopy, we show that negative supercoiling promotes local melting of duplex DNA at DNA-DNA crossings within braided molecules, generating single-stranded DNA that can be engaged by TOP3. These supercoiled substrates can be efficiently resolved by TOP3-RMI1-RMI2 (TRR), whereas relaxed DNA is resistant to TRR decatenation. Moreover, we find that RMI2 and the ssDNA binding Replication Protein A (RPA) stimulates TOP3 decatenation and demonstrate that decatenation remains viable up to 30pN of tension on the substrate. These findings reveal a mechanism by which torsional stress converts an otherwise inaccessible duplex DNA crossing into a substrate for a single-strand-specific topoisomerase. We propose that negative supercoiling can therefore direct type IA topoisomerases to topological DNA linkages by promoting local duplex melting, providing a physical mechanism that couples DNA supercoiling to the resolution of DNA entanglements.

molecular biology↗
bioRxiv2026

Molecular Mechanism of Condensin II auto-repression by NCAPD3 and activation by M18BP1

Human condensin II is a constitutively nuclear molecular motor that initiates chromosome organization in early mitosis. How condensin II is activated specifically in mitosis remains unknown. Here, we describe the molecular mechanism underlying condensin II auto-repression and activation. By determining multiple structural states of condensin II, we discovered that an autoinhibitory tail within the NCAPD3 subunit (NCAPD3Tail) holds the complex in a conformation that is incompatible with DNA capture. Deletion of NCAPD3Tail spontaneously activates condensin II in cells, illuminating its autoinhibitory role in vivo. We further show this translates to increased loop DNA formation by condensin II in vitro. Direct competition for the NCAPD3Tail binding site on condensin II enables the putative activator protein M18BP1 to liberate a key DNA-binding element in the NCAPH2 N-terminus, enabling DNA capture. Unexpectedly, M18BP1 not only relieves autoinhibition but also directly contributes to DNA organization by forming a positively charged loop that enhances DNA-anchoring by condensin II. Together, these findings reveal a bipartite activation mechanism wherein M18BP1 relieves autorepression, and renders condensin II biochemically competent to form stable DNA loops, ensuring highly stringent regulation of mitotic chromosome formation.

biochemistry↗
bioRxiv2024

Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent

Structural maintenance of chromosome (SMC) complexes organize and regulate genomes by extruding DNA loops. During loop extrusion, DNA can be reeled into the growing loop from one or both sides, generating distinct extrusion directionality states whose physical basis remains unclear. Here, we combine single-molecule analysis and molecular dynamics simulations to investigate loop extrusion directionality across SMC complexes. We show that dimeric Smc5/6 and Wadjet predominantly perform two-sided loop extrusion during initial loop growth, whereas monomeric condensin exhibits one-sided extrusion, consistent with a relationship between stoichiometry and extrusion directionality. Surprisingly, however, cohesin predominantly exhibits one-sided extrusion despite functioning as a dimeric complex. Notably, dimeric Smc5/6 and Wadjet progressively transition from two-sided to one-sided extrusion as loop growth matures. Simulations and force-dependent analysis reveal that loop extrusion directionality is governed by a tension-dependent transition in dimeric motors. Increasing DNA tension erodes two-sided extrusion near the motor stalling force, driving a tug-of-war regime in which competing motors transiently dominate one another. This transition promotes one-sided extrusion during mature Smc5/6 and Wadjet-mediated loop extrusion and places cohesin, which has a comparatively low stalling force ([~]0.1 pN), constitutively near this regime. Together, our findings establish loop extrusion directionality as a dynamic emergent property governed by SMC stoichiometry, DNA tension, and stochastic motor competition.

biophysics↗
bioRxiv2024

Molecular Mechanism of Condensin I Activation by KIF4A

During mitosis, the condensin I and II complexes compact chromatin into chromosomes. Loss of the chromokinesin, KIF4A, results in reduced condensin I chromosome association. However, the molecular mechanism behind this phenotype is unknown. Here, we show that KIF4A binds directly to condensin I HAWK subunit, NCAPG, via a conserved disordered short linear motif (SLiM) found in its C-terminal tail. KIF4A binding directly competes with two auto-inhibitory interactions in condensin I, mediated by SLiMs found in the N-terminus of NCAPH and the C-terminus of NCAPD2, which bind sites that overlap with KIF4A binding on NCAPG. Addition of the KIF4A SLiM peptide alone is sufficient to stimulate condensins ATPase and DNA loop extrusion activity. We also identify SLiMs in known yeast condensin interactors, Sgo and Lrs4, that bind yeast Ycg1, the HAWK equivalent to NCAPG. Our findings, together with previous work on condensin II and cohesin, demonstrate that SLiMs binding to HAWK subunits is a conserved mechanism of regulation in SMC complexes.

biochemistry↗
bioRxiv2023

Substrate accessibility regulation of human TopIIa decatenation by cohesin

Human topoisomerase II alpha (TOP2) is the main mitotic decatenase 1-3 resolving intertwines between sister chromatids that form during DNA replication 4. Here, we employ quadruple-trap optical tweezers to generate braids between a pair of {lambda}-DNA molecules to study, at the single-molecule level and in real-time, how TOP2 untangles these DNAs. We find that TOP2 rapidly resolves single and multiple braids and is inhibited by chemotherapy agent, etoposide. TOP2 is sensitive to DNA conformation, exhibiting a chiral preference for the removal of braids with right-handed crossings and is inhibited when DNAs are held at forces over 20pN. We show that TOP2 must load at the cross between the two DNAs to resolve braids efficiently. TOP2 pre-loaded onto individual DNAs is unable to resolve newly formed braids in the presence of ATP, suggesting that the geometry of pre-bound enzyme is incompatible with the capture of the second DNA. Finally, we show that cohesin binds to braids, preventing TOP2 from resolving them. Our study unveils novel insights into the regulation of TOP2s decatenation, underscoring the importance of substrate accessibility and the role of cohesin in modulating TOP2 activity.

biophysics↗
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↗
bioRxiv2021

Cohesin is essential for the timely activation of condensin and anaphase segregation

Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role in determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids are maintained during anaphase in the absence of cohesin. Here we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, TEV-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wildtype cells and show that they play a role in the organisation of centromeric regions. Our data demonstrate that in yeast cells, cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating. One sentence summaryCohesin complexes on yeast chromosomes provide organisation to segregating chromatids.

cell biology↗
bioRxiv2019

FACT mediates cohesin function on chromatin

Cohesin is a key regulator of genome architecture with roles in sister chromatid cohesion 1,2 and the organisation of higher-order structures during interphase 3 and mitosis 4,5. The recruitment and mobility of cohesin complexes on DNA is restricted by nucleosomes 6-8. Here we show that cohesin role in chromosome organisation requires the histone chaperone FACT. Depletion of FACT in metaphase cells affects cohesin stability on chromatin reducing its accumulation at pericentric regions and binding on chromosome arms. Using Hi-C, we show that cohesin-dependent TAD (Topological Associated Domains)-like structures in G1 and metaphase chromosomes are disrupted in the absence of FACT. Surprisingly, sister chromatid cohesion is intact in FACT-depleted cells, although chromosome segregation failure is observed. Our results uncover a role for FACT in genome organisation by facilitating cohesin-dependent compartmentalization of chromosomes into loop domains.

molecular biology↗
bioRxiv2019

A conserved activity for cohesin in bridging DNA molecules

Essential processes such as accurate chromosome segregation, regulation of gene expression and DNA repair rely on protein-mediated DNA tethering. Sister chromatid cohesion requires the SMC complex cohesin to act as a protein linker that holds replicated chromatids together (1, 2). The molecular mechanism by which cohesins hold sister chromatids has remained controversial. Here, we used a single molecule approach to visualise the activity of cohesin complexes as they hold DNA molecules. We describe a DNA bridging activity that requires ATP and is conserved from yeast to human cohesin. We show that cohesin can form two distinct classes of bridges at physiological conditions, a \"permanent bridge\" able to resists high force (over 80pN) and a \"reversible bridge\" that breaks at lower forces (5-40pN). Both classes of bridges require Scc2/Scc4 in addition to ATP. We demonstrate that bridge formation requires physical proximity of the DNA segments to be tethered and show that \"permanent\" cohesin bridges can move between two DNA molecules but cannot be removed from DNA when they occur in cis. This suggests that separate physical compartments in cohesin molecules are involved in the bridge. Finally, we show that cohesin tetramers, unlike condensin, cannot compact linear DNA molecules against low force, demonstrating that the core activity of cohesin tetramers is bridging DNA rather than compacting it. Our findings carry important implications for the understanding of the basic mechanisms behind cohesin-dependent establishment of sister chromatid cohesion and chromosome architecture.

biochemistry↗
bioRxiv2018

PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by the Mec1 kinase and its own auto-phosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 phosphorylation and activation has been well documented, less is known about the processes that counteract its kinase activity during the response to DNA damage. Here, we describe that PP4 phosphatase dephosphorylates Rad53 during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects DNA resection and repair by single-strand annealing, defects that are bypassed by reducing the hyper-phosphorylation state of Rad53 observed in the absence of the phosphatase. These results confirm that Rad53 is one of the main targets of PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection.

molecular biology↗