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Rueda, D. S.

Publications and source records attributed to Rueda, D. S..

13 recordsLinked to original sources
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

INO80 rapidly shuttles nucleosomes between chromatin barriers

ATP-dependent chromatin remodelers establish nucleosome organization, but how they move nucleosomes over extended distances and respond to neighboring chromatin remains unclear. Here, we combine correlative optical tweezers and fluorescence microscopy, and single-molecule FRET to visualize human INO80 activity on naked and chromatinized DNA. hINO80 undergoes free one-dimensional diffusion along DNA with multiple, nucleotide-regulated, diffusive states. Upon engaging a nucleosome, hINO80 drives rapid, processive nucleosome sliding over thousands of base pairs in bidirectional bursts. Neighboring nucleosomes prevent passage and redirect translocation, confining mobile nucleosomes to repeated movement between chromatin boundaries. Single-molecule FRET reveals transitions consistent with INO80 switching between opposing nucleosome-binding orientations, while fluorescence stoichiometry and mass photometry show that two hINO80 complexes can simultaneously occupy one nucleosome. These findings identify non-exclusive mechanisms for directional reversal and reveal how long-range hINO80 translocation is converted into boundary-constrained nucleosome repositioning, providing a dynamic framework for understanding nucleosome organization within chromatin.

biophysics↗
bioRxiv2026

Individual mouse mitotic chromosomes exhibit cell type-specific differences in biomechanical properties

Cyclic episodes of chromosome compaction and de-condensation are features of eukaryotic cell division that aid mitotic segregation and help prevent aneuploidy. While biophysical data on mitotic chromosome structure has been previously obtained, heterogeneity within samples can confound analyses and precludes direct like-for-like comparisons. To circumvent this, we employed advanced flow cytometry to purify specific metaphase chromosomes with biotinylated telomeres from stably engineered mouse cells. We show that ESC-derived metaphase chromosomes 3 and 19 display distinct properties but share a conserved force-dependent mechanical response. In contrast, chromosome equivalents isolated from NSCs and preB cells show markedly different force-dependent responses, reflecting progressive differentiation stages. Covalent crosslinking of ESC-derived chromosomes alters biomechanical properties to mimic equivalents from more differentiated cells. Collectively, these results highlight the need to isolate specific, homogeneous metaphase chromosome samples to accurately decipher their complex behaviours.

biophysics↗
bioRxiv2026

DeepDiffusion: a Physics-Informed Neural Network for Heterogeneous Facilitated 1D Diffusion

Single-molecule fluorescence microscopy combined with optical tweezers has enabled the direct observation of diffusing proteins on tethered DNA. These and complementary techniques reveal facilitated one-dimensional diffusion as a common functional mechanism for numerous DNA-binding proteins, with a wide range of heterogeneous diffusive behaviours arising from different DNA-binding modes. However, detailed investigations have been limited by the lack of methods to detect such heterogeneous diffusion. We have developed DeepDiffusion, a physics-informed neural network model for estimating the instantaneous diffusion at each point along a single-molecule trajectory. We show, using synthetic trajectories, that DeepDiffusion can accurately detect subtle changes in diffusion even when challenged with large underlying errors. DeepDiffusion can recapitulate previously characterised heterogeneity in experimental data and reveal mechanistic details of facilitated diffusion that are inaccessible to current methods. We expect DeepDiffusion to become a powerful tool for single-molecule researchers, allowing them to investigate the diffusion of their proteins of interest in unprecedented detail.

biophysics↗
bioRxiv2025

The ultrafine-bridge-associated endonuclease ANKLE1 is stimulated by tension in DNA to process branch-points

Covalent linkages between chromosomes are naturally formed as a result of recombination or DNA replication. These can persist until late mitosis, resulting in interchromosomal ultrafine bridges, preventing cell division and causing genome instability. The endonuclease ANKLE1, localised at the cell midbody during cytokinesis and selective for DNA branchpoints, is ideally poised to act as an enzyme of last resort to process interchromosomal bridges, enabling cell division to proceed. However, how ANKLE1 cleaves DNA under the tension existing in interchromosomal bridges during mitosis remains unexplored. Using optical tweezers, we show that ANKLE1 is a tension-stimulated endonuclease. High tension in DNA increases the ANKLE1 junction cleavage rate, with a twenty-fold increase at 60 pN. This indicates that ANKLE1 has evolved to respond to tension-induced DNA structural changes, thereby facilitating nucleolytic activity. This novel mechano-enzymological response of ANKLE1 reveals how it is well-suited to process ultrafine-bridges during late mitosis.

biophysics↗
bioRxiv2024

Herpes simplex virus type 1 origin binding protein UL9 tethers and loops origin- and non-origin-DNA intra- and intermolecularly

Herpesviruses are ubiquitous human pathogens, which are the causative agent of mild to severe symptoms ranging from cold sore to nasopharyngeal carcinoma. Even though replication of the linear dsDNA genome has been studied for decades, we still lack a complete molecular understanding of its mechanism. It has been proposed, but never shown directly, that the HSV-1 origin binding protein UL9 binds two closely located binding sites within the oriS origin sequence, thereby mediating origin looping, which in turn facilitates replication initiation. Here, we used an array of single-molecule approaches to test this long-standing hypothesis directly. Surprisingly, the data show that UL9 does not loop oriS efficiently. However, we demonstrate that UL9 can form large DNA loops at non-origin sequences very efficiently, as well as tether two oriS DNA molecules intermolecularly. Contrary to the origin bending hypothesis, our findings indicate that UL9 does not loop oriS DNA, but rather may play an alternative role in replication initiation, such as tethering two separate molecules to facilitate recombination.

microbiology↗
bioRxiv2024

Conserved helical motifs in the Ikaros IDR mediate NuRD interaction and transcriptional repression

The transcription factor IKZF1/Ikaros is essential for B cell development, and recurrently mutated in human B-ALL. Ikaros has been ascribed both activating and repressive functions via interactions with coactivator and corepressor complexes, but the relative abundance of Ikaros-associated coregulatory complexes and their contribution to Ikaros-mediated gene regulation are not well understood. To address this issue, we performed an unbiased identification of Ikaros-interacting proteins in pre-B cells, and found that Ikaros interacts overwhelmingly with corepressors and heterochromatin-associated proteins. Time-resolved analysis of transcription and chromatin state identified transcriptional repression as the immediate response to Ikaros induction. Transcriptional repression preceded transcriptional activation by several hours, and was accompanied by a rapid loss of chromatin accessibility and reduced levels of H3K27ac particularly at enhancers. Functional characterisation of intrinsically disordered regions in the Ikaros protein identified highly conserved helical motifs that mediate Ikaros association with the NuRD corepressor complex and contribute to the silencing of target genes in pre-B cells and antiproliferative functions of Ikaros in human B-ALL.

molecular biology↗
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↗
bioRxiv2023

The transcriptome of HTLV-1-infected primary cells following reactivation reveals changes to host gene expression central to the proviral life cycle

Infections by Human T cell Leukaemia Virus type 1 (HTLV-1) persist for the lifetime of the host by integrating into the genome of CD4+ T cells. Proviral gene expression is core to proviral survival and the maintenance of the proviral load, through the pro-proliferative changes it induces in infected cells. Despite their role in HTLV-1 infection and a persistent cytotoxic T lymphocyte response raised against them, proviral transcripts from the sense-strand are rarely detected in fresh cells extracted from the peripheral blood, and have recently been found to be expressed intermittently by a small subset of cells at a given time. Ex vivo culture of infected cells prompts synchronised proviral expression in infected cells from peripheral blood, allowing the study of factors involved in reactivation in primary cells. Here, we used bulk RNA-seq to examine the host transcriptome over six days in vitro, following proviral reactivation in primary peripheral CD4+ T cells isolated from subjects with non-malignant HTLV-1 infection. Infected cells displayed a conserved response to reactivation, characterised by discrete stages of gene expression, cell division and subsequently horizontal transmission of the virus. We observed widespread changes in Polycomb gene expression following reactivation, including an increase in PRC2 transcript levels and diverse changes in the expression of PRC1 components. We hypothesize that these transcriptional changes constitute a negative feedback loop that maintains proviral latency by re-deposition of H2AK119ub1 following the end of proviral expression. Using RNAi, we found that certain deubiquitinases, BAP1, USP14 and OTUD5 each promote proviral transcription. These data demonstrate the detailed trajectory of HTLV-1 proviral reactivation in primary HTLV-1-carrier lymphocytes and the impact on the host cell. Author summaryHuman T cell Leukemia Virus type I (HTLV-1) is a retrovirus which causes an aggressive leukemia or lymphoma, or a chronic inflammatory disease of the central nervous system, in a subset ({approx} 10%) of affected carriers. Whilst the virus is only intermittently expressed by infected cells, the virus persists in its host by increasing the proliferation rate and survival of infected cells. It is therefore imperative to understand the mechanisms that control the activation and deactivation of the virus. We examined the expression of host and viral genes during HTLV-1 reactivation in cells freshly isolated from patients blood. The infected cells displayed consistent changes in gene expression over six days. We observed changes in the expression of the Polycomb group of epigenetic modifiers, known to impact HTLV-1 transcription, which may form a negative-feedback mechanism allowing the virus to return to a latent (quiescent) state following activation. We additionally identified a set of three deubiquitinases that increase expression of the virus. Our data detail the changes in gene expression underlying core aspects of the HTLV-1 life cycle in primary cells, and provide a resource for further investigation.

microbiology↗
bioRxiv2020

Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins

Homologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, while RFS-1/RIP-1 acts as a chaperone to promote 3 to 5 filament growth via highly dynamic engagement with 5 filament ends. Inhibiting ATPase or mutation in RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

biophysics↗
bioRxiv2019

Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding ({beta}-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

cell biology↗
bioRxiv2019

Retroviral integration into nucleosomes throughDNA looping and sliding along the histone octamer

Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 [A] resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Forster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities with chromatin remodelers.

biochemistry↗