Biology subjects

Britton, S.

Publications and source records attributed to Britton, S..

15 recordsLinked to original sources

bioRxiv2026

Sequential division of labor between PAXX and XLF drives NHEJ synaptic complex stability and maturation

DNA double-strand breaks (DSBs) are primarily repaired by non-homologous end joining (NHEJ), which tethers and ligates DNA ends within a synaptic complex. PAXX is an NHEJ accessory factor related to XRCC4 and XLF, but its mechanistic role and genetic interaction with XLF are unclear. Using biochemical assays and single-molecule imaging within Xenopus laevis egg extracts and human cell assays, we show that PAXX bridges DNA ends by using its disordered tails to bind opposing Ku molecules. This PAXX tether substantially extends long-range synaptic complex (LRSC) lifetime and modestly stabilizes the short-range synaptic complex (SRSC). LRSC stabilization increases XLF residence and promotes the LRSC-to-SRSC transition. Using domain-swapped chimeras, we demonstrate that PAXXs tails provide synaptic stabilization, whereas XLFs heads interact with XRCC4 to drive the LRSC-to-SRSC transition: an XLF head-PAXX tail chimera recapitulates the functions of both proteins, revealing complementary, sequential roles for PAXX and XLF in NHEJ.

biophysics↗

bioRxiv2026

Exploiting HSD17B11-dependent dialkynylcarbinols cytotoxicity for facile CRISPR/Cas9-based gene inactivation

Several approaches have been developed to improve the efficiency of CRISPR/Cas9-based genome editing, including the co-inactivation of a gene whose loss confers resistance to a cytotoxic compound, thereby enabling enrichment of successfully edited cells. Here, we show across multiple cell lines that inactivation of HSD17B11, a non-essential member of the Short-chain Dehydrogenase/Reductase (SDR) superfamily, confers strong resistance (29- to 131-fold) to a Phenyl diAlkynylCarbinol compound (PAC) in both human and mouse cells, without affecting cell viability or proliferation. We demonstrate that co-inactivation of HSD17B11 followed by PAC selection can be used to rapidly identify efficient guide RNAs targeting a gene of interest and to readily isolate clones inactivated for one or multiple genes. Altogether, these results establish a simple and efficient experimental strategy for generating knockout cells by using PAC selection to enrich for successfully edited cells.

molecular biology↗

bioRxiv2026

Selective Disruption of Plasmodium falciparum mitochondrial DNA via G-Quadruplex-Binding Ligand RHPS4 Provides a Novel Antimalarial Strategy

Malaria caused by Plasmodium falciparum remains a major health threat, killing over 600,000 people annually. The spread of resistance to all major antimalarials, including artemisinins, highlights the urgent need for new drugs with distinct mechanisms of action. Here we show that the G-quadruplex ligand RHPS4, an acridine derivative, displays strong antiplasmodial activity against both drug-sensitive and -resistant P. falciparum strains and clinical isolates. RHPS4 primarily targets the trophozoite stage and induces major mitochondrial alterations, including reduction of mitochondrial DNA (mtDNA) and transcriptional dysfunctions. Bioinformatic analyses identified at least eight putative G4-forming sequences within the parasites mtDNA. Biophysical studies confirmed G4 folding of at least one sequence and its interaction with RHPS4. These findings indicate that RHPS4 disrupts P. falciparum mitochondrial metabolism through G4 stabilization, leading to parasite death, and establish mtDNA G4 structures as novel therapeutic targets for antimalarial development.

microbiology↗

bioRxiv2025

Antimicrobial stewardship in animal health: experience-based co-design framework to capture progress and accelerate improvement

Antimicrobial stewardship (AMS) is essential for mitigating antimicrobial resistance (AMR) in animal health but implementing national initiatives is challenging due to diverse influencing factors and the need for context-specific approaches. An Animal AMS Practice Group, comprised of individuals with lived experience overseeing AMS in various animal health contexts was engaged, and through an experience-based co-design approach created a comprehensive AMS Framework that captures progress and supports tangible improvements in AMS practices in each context. The Framework supported a cross-sectoral pilot assessment that helped users identify progress, areas for improvement, and contextualise antimicrobial usage and AMR data, while also motivating further AMS efforts. Despite common barriers to sharing sensitive data, participants willingly shared AMS results for benchmarking and publication. The process demonstrated that co-design coupled with peer learning and expert support are essential to ensuring AMS tools are utilised in practice. Several participants incorporated the Framework into routine practice, with some using it to drive sector-level AMS action. The resulting Framework offers an adaptable, scalable entry point for AMS efforts and a platform for setting meaningful improvement goals. It supports broader opportunities for national-level AMS assessments and strategy development. Impact StatementThis research offers a practical, scalable approach to improving AMS in animal health. By embedding behaviour change into the project through an experience-based co-design process, rather than expecting it to occur afterward, veterinarians, animal managers, and other frontline professionals defined what good AMS looks like in their context and identified achievable actions to improve daily practices that aligned across diverse animal health settings. The resulting AMS Framework provides a clear, accessible starting point for those overseeing daily AMS activities to benchmark practices, set meaningful goals, and track progress, while also informing national-level prioritisation and decision-making. This user-led model demonstrates that when AMS tools are co-developed with those who will use them and users are empowered to shape the projects design and outputs, the resulting tools are more likely to be adopted, shared, and sustained.

microbiology↗

bioRxiv2025

Patching up the nucleus: a novel role for PMLII in nuclear envelope rupture repair

The nuclear envelope (NE) is important for cellular health as it protects and organizes the genome. NE dynamics is important for various cellular processes including cell growth, migration and removal of defective NE components. In extreme cases, the NE can rupture leading to exchange of material between the nuclear interior and the cytoplasm. Rapid repair of the NE is initiated to minimize the effect on the genome. While our understanding of the machinery involved in this repair process is increasing, a lot is still unknown about this process including events leading up to NE rupture. Interestingly, biomolecular condensates have recently been found to play important roles in membrane repair and remodelling in cells. Here, we found that promyelocytic leukemia protein isoform II (PMLII), a protein involved in nuclear PML body formation, forms condensates at the NE. These condensates specifically form at sites where the lamina is disrupted. We show that NE rupture often occurs at these sites and that PMLII stays present until rupture repair is initiated suggesting a role in stabilization of the site for effective repair.

cell biology↗

bioRxiv2024

DNA polymerase Lambda is anchored within the NHEJ synaptic complex via Ku70/80

Non-homologous end joining (NHEJ) is the predominant pathway by which double-strand DNA breaks (DSBs) are repaired in mammals. To enable final break closure, various NHEJ end-processing factors respond to the chemistry of the damaged DNA ends. Amongst these factors is DNA polymerase lambda (Pol {lambda}), a member of the Pol X family. How members of the Pol X family engage with the NHEJ complex is unknown. Here, we present cryo-EM structures of Pol {lambda} in complex with the Ku70/80 DSB sensor whilst engaged with the DNA-PK holoenzyme in a long-range synaptic complex. These structures reveal a specific interaction site between Ku70/80 and the Pol {lambda} BRCT domain. The functionality of this interaction is assessed by generating point mutations on either side of the Pol {lambda} BRCT:Ku70/80 interface. Using these mutants in two orthogonal assays in cells (live protein recruitment at biphoton laser-damaged nuclear sites and transfection with an original gap-filling reporter plasmid) defines the molecular basis and essentiality of the BRCT domain for the recruitment and activity of the Pol {lambda} within the NHEJ complex. Ultimately, these data explain the role of this interaction in cell survival to DSBs. Finally, we propose a unified model for the interaction of the three Pol X family members bearing BRCT domains with the same site of Ku70/80.

biochemistry↗

bioRxiv2024

Identification of the main barriers to Ku accumulation in chromatin

Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to [~]1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions. HighlightsO_LIDNA-PKcs structurally blocks Ku sliding into chromatin in human & Xenopus C_LIO_LIA neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin C_LIO_LIIn S-phase, ATM/CtIP overcomes Ku accumulation C_LIO_LIIn absence of DNA-PKcs, transcription at the DNA end vicinity is inhibited C_LI eTOC blurbThe DNA end binding protein Ku can slide onto naked DNA but this is limited in cells. Using human cells and Xenopus egg extracts, DNA-PKcs is identified as the main structural barrier to Ku entry into chromatin, along with two active mechanisms which limit Ku accumulation in absence of DNA-PKcs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/574002v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@4ebc45org.highwire.dtl.DTLVardef@12a7805org.highwire.dtl.DTLVardef@12db0a8org.highwire.dtl.DTLVardef@a925cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Characterisation of Plasmodium vivax lactate dehydrogenase dynamics in P. vivax infections

Plasmodium vivax lactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway of Plasmodium vivax. It can also be used as a diagnostic biomarker. Quantitation of plasma PvLDH has been used as a measure of P. vivax biomass in clinical studies of uncomplicated and severe vivax malaria. With the increasing importance of PvLDH in studying P. vivax diagnosis and infection, improved characterisation of the dynamics of this biomarker is important. In this study, we developed mathematical models that capture parasite and matrix PvLDH dynamics in ex vivo culture and the human host. We estimated the biological parameters using ex vivo and in vivo longitudinal data of parasitemia and PvLDH concentration collected from P. vivax-infected humans using Bayesian hierarchical inference. We found that the ex vivo and in vivo estimates of PvLDH in a parasitized red blood cell differed significantly across the asexual life cycle, with in vivo estimates at least ten-fold higher than ex vivo estimates (for example, the median estimate of intraerythrocytic PvLDH mass at the end of the life cycle was 9.4x10-3 ng in vivo vs. 5.1x10-4 ng ex vivo). We also estimated the ex vivo PvLDH half-life to be 65.3 h (95% credible interval: 60.8--70.7 h), which is approximately three times longer than the median estimate of the in vivo PvLDH half-life, 21.9 h (16.7--29.9 h). Our findings provide an important foundation to further improve quantitative understanding of P. vivax biology and facilitate the development of PvLDH-based diagnostic tools.

biophysics↗

bioRxiv2022

A Chemical-mechanical Coupled Model Predicts Roles of Spatial Distribution of Morphogen in Maintaining Tissue Growth

The exact mechanism controlling cell growth remains a grand challenge in developmental biology and regenerative medicine. The Drosophila wing disc tissue serves as an ideal biological model to study growth regulation due to similar features observed in other developmental systems. The mechanism of growth regulation in the wing disc remains a subject of intense debate. Most existing models to study tissue growth focus on either chemical signals or mechanical forces only. Here we developed a multiscale chemical-mechanical coupled model to test a growth regulation mechanism depending on the spatial range of the morphogen gradient. By comparing the spatial distribution of cell division and the overall shape of tissue obtained in the coupled model with experimental data, our results show that the distribution of the Dpp morphogen can be critical in resulting tissue size and shape. A larger tissue size with a faster growth rate and more symmetric shape can be achieved if the Dpp gradient spreads in a larger domain. Together with the absorbing boundary conditions, the feedback regulation that downregulates Dpp receptors on the cell membrane allows the further spread of the morphogen away from its source region, resulting in prolonged tissue growth at a more spatially homogeneous growth rate. Summary StatementA multiscale chemical-mechanical model was developed by coupling submodels representing dynamics of a morphogen gradient at the tissue level, intracellular chemical signals, and mechanical properties at the subcellular level. By applying this model to study the Drosophila wing disc, it was found that the spatial range of the morphogen gradient affected tissue growth in terms of the growth rate and the overall shape.

developmental biology↗

bioRxiv2021

SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/460423v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@12daae1org.highwire.dtl.DTLVardef@12db406org.highwire.dtl.DTLVardef@1863517org.highwire.dtl.DTLVardef@7b548_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

biochemistry↗

bioRxiv2021

Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

biochemistry↗

bioRxiv2020

Role of combined cell membrane and wall mechanicalproperties regulated by polarity signals in cell budding

The budding yeast, Saccharomyces cerevisiae, is a prime biological model to study mechanisms underlying asymmetric growth. Previous studies have shown that, prior to yeast bud emergence, polarization of a conserved small GTPase, Cdc42, must be established. Additionally, hydrolase changes the mechanical properties of the cell wall and plasma membrane with the periplasm between them (cell surface). However, how the surface mechanical properties in the emerging bud are different from the properties of the mother cell and their role in bud formation are not well understood. We hypothesize that the polarized chemical signal alters the local dimensionless ratio of stretching to bending stiffness of the cell surface of the emerging yeast bud. To test this hypothesis, a novel three-dimensional coarse-grained particle-based model has been developed which describes inhomogeneous mechanical properties of the cell surface. Model simulations suggest that regulation of the dimensionless ratio of stretching to bending stiffness of the cell surface is necessary to initiate bud formation. Furthermore, model simulations predict that bud shape depends strongly on the experimentally observed molecular distribution of the polarized signaling molecule Cdc42, while the neck shape of the emerging bud is strongly impacted by the properties of the chitin and septin ring. This 3D model of asymmetric cell growth can also be used for studying viral budding and other vegetative reproduction processes performed via budding.

biophysics↗

bioRxiv2020

XAB2 prevents abortive recombinational repair of replication-associated DNA double-strand breaks and its loss is synthetic lethal with RAD52 inhibition

Unrepaired O6-methylguanine lesions induced by the alkylating chemotherapy agent temozolomide lead to replication-associated single-ended DNA double-strand breaks (seDSBs) that are repaired predominantly through RAD51-mediated homologous recombination (HR). Here, we show that loss of the pre-mRNA splicing and DNA repair protein XAB2 leads to increased temozolomide sensitivity in glioblastoma cells, which reflects abortive HR due to Ku retention on resected seDSBs. XAB2-dependent Ku eviction also occurred at seDSBs generated by the topoisomerase I poison campthotecin and operated in parallel to an ATM-dependent pathway previously described. Although Ku retention elicited by loss of XAB2 did not prevent RAD51 focus formation, the resulting RAD51-ssDNA associations were unproductive, leading to increased engagement of non-homologous-end-joining in S/G2 and genetic instability. Overexpression of RAD51 or the single-stranded DNA annealing factor RAD52 rescued the XAB2 defects. RAD52 depletion led to severe temozolomide sensitivity, whereas a synthetic lethality interaction was observed between RAD52 and XAB2.

molecular biology↗

bioRxiv2020

Machine Learning and Optimal Control of Enzyme Activities to Preserve Solvent Capacity in the Cell

Experimental measurements or computational model predictions of the post-translational regulation of enzymes needed in a metabolic pathway is a difficult problem. Consequently, regulation is mostly known only for well-studied reactions of central metabolism in various model organisms. In this study, we utilize two approaches to predict enzyme regulation policies and investigate the hypothesis that regulation is driven by the need to maintain the solvent capacity in the cell. The first predictive method uses a statistical thermodynamics and metabolic control theory framework while the second method is performed using a hybrid optimization-reinforcement learning approach. Efficient regulation schemes were learned from experimental data that either agree with theoretical calculations or result in a higher cell fitness using maximum useful work as a metric. Model predictions provide the following novel general principles: (1) the regulation itself causes the reactions to be much further from equilibrium instead of the common assumption that highly non-equilibrium reactions are the targets for regulation; (2) regulation is used to maintain the concentrations of both immediate and downstream product concentrations rather than to maintain a specific energy charge; and (3) the minimal regulation needed to maintain metabolite levels at physiological concentrations also results in the maximal energy production rate that can be obtained at physiological conditions. The resulting energy production rate is an emergent property of regulation which may be represented by a high value of the adenylate energy charge. In addition, the predictions demonstrate that the amount of regulation needed can be minimized if it is applied at the beginning or branch point of a pathway, in agreement with common notions. The approach is demonstrated for three pathways in the central metabolism of E. coli (gluconeogenesis, glycolysis-TCA and Pentose Phosphate-TCA) that each require different regulation schemes. It is shown quantitatively that hexokinase, glucose 6-phosphate dehydrogenase and glyceraldehyde phosphate dehydrogenase, all branch points of pathways, play the largest roles in regulating central metabolism.

systems biology↗

bioRxiv2020

Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands

G-quadruplexes (G4), non-canonical DNA structures, are involved in several essential processes. Stabilization of G4 structures by small compounds (G4 ligands) affects almost all DNA transactions, including telomere maintenance and genomic stability. Here, thanks to a powerful and unbiased genetic approach, we identify topoisomerase 2-alpha (TOP2A) as the main effector of cell cytotoxicity induced by CX5461, a G4 ligand currently undergoing phase I/II clinical trials. This approach also allowed to identify new point mutations affecting TOP2A activity without compromising cell viability. Moreover, based on cross-resistance studies and siRNA-based protein depletion we report that TOP2A plays a major role in cell cytotoxicity induced by two unrelated clastogenic G4 ligands, CX5461 and pyridostatin (PDS). We also report that cytotoxic effects induced by both compounds are associated with topoisomerase 2-mediated DNA breaks production. Finally, we show that TOP2-mediated DNA breaks production is strongly associated with RNA Pol II-dependent transcription and is countered by topoisomerase 1 (TOP1). Altogether our results indicate that clastogenic G4 ligands act as DNA structure-driven TOP2-poisons at transcribed regions bearing G-quadruplex structures.

molecular biology↗