Biology subjects

Pyne, A. L. B.

Publications and source records attributed to Pyne, A. L. B..

13 recordsLinked to original sources

bioRxiv2026

TRF2 mediates force-resistant bridging of distal telomeric DNA repeats

Shelterin binds telomeres, regulates telomere length, and protects chromosome ends from mis-repair, which is essential for genome stability. Telomere end-protection uniquely depends on the TRF2 subunit, which assembles telomeric DNA into a protective T-loop structure in vivo, yet the biochemical mechanism underlying this process remains unresolved. Using single-molecule optical tweezers and atomic force microscopy, we show that the human shelterin complex tethers distal telomeric DNA repeats, looping the intervening DNA. These bridges withstand tens of piconewtons of applied load, rupture at a force that scales with telomeric repeat number and re-form on relaxation. Structure-function analysis demonstrates that this force-resistant tethering is TRF2-dependent under our conditions and depends on multiple interfaces, with the TRFH domain making a major but non-exclusive contribution: introducing the TRF2 TRFH domain into TRF1 is sufficient to confer tethering, whereas its deletion from TRF2 reduces but does not abolish activity. We propose telomeric dsDNA tethering by TRF2 as a candidate first step in T-loop formation, preceding invasion of the telomeric 3' ssDNA overhang.

biophysics↗

bioRxiv2026

Deep Learning-Enhanced TopoStats for the Automated Quantification of DNA and Complex Biomolecular Structures

Atomic force microscopy (AFM) enables nanometre-scale, label-free imaging of biomolecules and surfaces under near-native conditions, yet quantitative analysis of AFM data remains limited compared to other bioimaging modalities. This limitation largely arises from the absence of open, automated tools capable of addressing AFM-specific artefacts, data formats, and topographical outputs. Here, we present the latest version of TopoStats, an open-source Python package for automated and quantitative AFM image analysis, developed as a deep-learning enabled advancement of our original TopoStats software to support more complex samples and richer molecular characterisation. The pipeline integrates all key processing stages, including image flattening and noise correction, object detection and segmentation, morphometric feature extraction, and strand tracing with topological classification. Designed for accessibility and reproducibility, TopoStats adheres to the FAIR for Research Software (FAIR4RS) principles and provides configurable workflows adaptable to diverse biological samples. Combining high-resolution AFM and our analysis pipeline allows the quantification of subtle structural changes within a heterogeneous sample set, revealing properties not accessible with other structural biology techniques. We demonstrate the effectiveness of our pipeline to differentiate between plasmids with both different topology and sequence, by extracting meaningful quantitative descriptors that distinguish the samples with statistical significance. Collectively, these developments establish TopoStats as a versatile framework for high-throughput, quantitative AFM analysis, advancing AFM from a fundamentally qualitative visualisation technique toward a quantitative analytical tool.

biophysics↗

bioRxiv2025

Imaging and mechanism of DNA-DNA recognition mediated by divalent ions

In the cell, DNA must be tightly packed to facilitate its organisation into the nucleus, where recognition of homologous sequences underpins key processes such as recombination. Yet the structural basis of DNA-DNA pairing remains unknown. Here we combine high-resolution AFM and atomistic MD simulations to provide the first direct visualisation of DNA pairing in the presence of divalent ions. We show that strongly paired DNAs often achieve groove-to-groove alignment, driven by ionic bridges connecting the minor grooves of the two duplexes. These contacts are further stabilized by sequence-specific interactions, whose strength and specificity vary with the ion type. This mechanism of ion-mediated groove alignment provides a molecular framework for the long-hypothesized "helical alignment" model, in which homologous recognition is facilitated by preserving structural register between the two helices. Together, our findings reveal a fundamental principle by which divalent ions promote DNA-DNA pairing, with broad implications for chromosomal condensation and genome organization.

biophysics↗

bioRxiv2025

Cytosolic DNA structures produced by mismatch-repair deficiency coordinate anti-tumor immunity in colorectal cancer

Patients with the microsatellite instable (MSI) subtype of colorectal cancer (CRC) have better prognosis and immunotherapy response than patients with the chromosomally instable (CIN) subtype due to improved cytotoxic T cell responses from high neoantigen levels and production of the chemokines CXCL10 and CCL5 that recruit cytotoxic T cells. This high chemokine production in MSI CRCs is due to constitutive activation of the cytosolic DNA (cyDNA) sensor STING by specific features of MSI cyDNA that lead to more effective STING pathway activation. Here, we investigate the features of MSI and CIN cyDNA to identify structures that more effectively activate STING. We find that MSI cyDNA is enriched in G-quadruplexes which improve STING and CD8+ T cell activation. Additionally, MSI micronuclei are also more efficient at inducing chemokine expression than CIN micronuclei. However, micronuclei are less effective than free cyDNA at inducing anti-tumor immunity and instead lead to increased Treg activation and IL10 production. Overall, these data highlight the role of specific cyDNA structures in anti-tumor immunity and provide essential knowledge for improved design of therapeutic DNA-based STING agonists that could be combined with immune checkpoint therapies to improve the prognosis of poorly immunogenic tumors like CIN CRCs.

cancer biology↗

bioRxiv2025

Protein-mediated stabilization and nicking of the non-template DNA strand dramatically affect R-loop formation in vitro

R-loops are an important class of non-B DNA structures that form co-transcriptionally. Using in vitro transcription and unbiased quantitative sequencing readouts, we show that the addition of single-strand DNA binding proteins co-transcriptionally can drive a 3- to 5-fold increase of R-loop frequency without significant changes to R-loop distribution. We propose that this is caused by stabilizing and preventing the collapse of short nascent R-loops. This suggests that R-loop formation is highly dynamic and highlights single strand binding proteins as players in cellular R-loop regulation. We further show that non-template strand DNA nicks are powerful initiators of R-loop formation, increasing R-loop frequencies by up to two orders of magnitude. Atomic force microscopy (AFM) revealed that the non-template strand in nick-initiated structures is often flayed away from the RNA:DNA hybrid and engaged in self-pairing, creating unique forked R-loop features. DNA nicks, one of the most frequent DNA lesions in cells, are therefore potential hotspots for opportunistic R-loop initiation and may cause the formation of a novel class of R-loops. Overall, this work highlights the importance of the displaced single-strand on R-loop initiation and dynamics. SIGNIFICANCE STATEMENTR-loops are three-stranded DNA:RNA hybrid structures that form during transcription and play critical roles in both gene regulation and genome stability. This study reveals that single-stranded DNA-binding protein (SSB) increases R-loop frequency by stabilizing nascent R-loops, revealing a role for this protein in R-loop dynamics. Additionally, single strand DNA breaks, common cellular lesions, are identified as potent initiators of R-loops, increasing their rate of formation by up to 100-fold. Using atomic force microscopy, the study uncovers unique forked structures in nick-initiated R-loops, representing a novel class of secondary R-loop structures. These findings provide new insights into how cellular factors and DNA damage influence R-loop formation, with implications for understanding genome instability in health and disease.

molecular biology↗

bioRxiv2024

Multiscale topological analysis of kinetoplast DNA via high-resolution AFM

Kinetoplast DNA is a complex nanoscale network, naturally assembled from thousands of interconnected DNA circles within the mitochondrion of certain parasites. Despite the relevance of this molecule to parasitology and the recent discovery of tuneable mechanics, its topology remains highly contested. Here we present a multiscale analysis into the structure of kDNA using a combination of high-resolution atomic force microscopy and custom-designed image analysis protocols. By capturing a notably large set of high-resolution images, we are able to look beyond individual kDNA variations and quantify population properties throughout several length scales. Within the sample, geometric fluctuations of area and mean curvature are observed, corresponding with previous in-vitro measurements. These translate to localised variations in density, with a sample-wide decrease in DNA density from the outer rim of the molecule to the centre and an increase in pore size. Nodes were investigated in a single molecule study, and their estimated connectivity significantly exceeded mean valence, with a high dependence on their position in the network. While node separation was approximately half the minicircle circumference, it followed a strong bimodal distribution, suggesting more complex underlying behaviour. Finally, upon selective digestion of the network, breakdown of the fibril-cap heterogeneity was observed, with molecules expanding less upon immobilisation on the mica surface. Additionally, selective digestion was seen in localised areas of the network, increasing pore size disproportionately. Overall, the combination of high-resolution AFM and single molecule image analysis provides a promising method to the continued investigation of complex nanoscale structures. These findings support the ongoing characterisation of kDNA topology to aid understanding of its biological and mechanical phenomena.

biophysics↗

bioRxiv2024

Under or Over? Tracing Complex DNA Topologies with High-Resolution Atomic Force Microscopy

The topology of DNA plays a crucial role in the regulation of cellular processes and genome stability. Despite its significance, DNA topology remains challenging to determine due to the length and conformational complexity of individual topologically constrained DNA molecules. We demonstrate unparalleled resolution of complex DNA topologies using Atomic Force Microscopy (AFM) in aqueous conditions. We present a new high-throughput automated pipeline to determine DNA topology from raw AFM images, using deep-learning methods to trace the backbone of individual DNA molecules and identify crossing points. Our pipeline efficiently determines which segment passes over which, including the handling of challenging crossings, where the path of each molecule may be harder to resolve. We demonstrate the wider applicability of our tracing method by determining the structure of stalled replication intermediates from Xenopus egg extracts, including theta structures and late replication products. By developing new methodologies to accurately trace the DNA path through every crossing, we determine the topology of plasmids, knots and catenanes from the E. coli Xer recombination system. In doing so we uncover a recurrent depositional effect and reveal its origins using coarse-grained simulations. Our approach is broadly applicable to a range of nucleic acid structures, including those which interact with proteins, and opens avenues for understanding fundamental biological processes which are regulated by or affect DNA topology.

biophysics↗

bioRxiv2023

Complement-mediated killing of bacteria by mechanical destabilization of the cell envelope

Complement proteins eliminate Gram-negative bacteria in serum via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. Initially, bacteria survived despite the formation of hundreds of MACs randomly distributed over the cell surface. This was followed by larger-scale disruption of the outer membrane, including propagating defects and fractures, and by an overall swelling and stiffening of the bacterial surface, which precede inner membrane permeation. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, causing inner membrane permeation and cell death. This represents a previously unknown route to bacterial cell death that could be exploited by novel antibiotic treatments.

microbiology↗

bioRxiv2022

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

biophysics↗

bioRxiv2020

TopoStats - an automated tracing program for AFM images

We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, 90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub (https://github.com/afm-spm/TopoStats) and is intended to be modified and adapted for use if required.

biophysics↗

bioRxiv2019

Combining high-resolution AFM with MD simulations shows that DNA supercoiling induces kinks and defects that enhance flexibility and recognition.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

biophysics↗

bioRxiv2019

Rapid antimicrobial sensitivity testing by single cell nanoscale optical interference

Growing antimicrobial resistance (AMR) is a serious global threat to human health. Current methods to detect resistance include phenotypic antibiotic sensitivity testing (AST) which measures bacterial growth and is therefore hampered by slow time to result (~12-24 hours). Therefore new rapid phenotypic methods for AST are urgently needed. Nanomechanical cantilever sensors have recently shown promise for rapid AST but challenges of bacterial immobilization can lead to variable results. Herein a novel cantilever-based method is described for detecting phenotypic antibiotic resistance within ~45 minutes, capable of detecting single bacteria. This method does not require complex, variable bacterial immobilization, and instead uses the laser and detector system to detect single bacterial cells in media as they pass through the laser focus. This provides a simple read out of bacterial antibiotic resistance by detecting growth (resistant) or death (sensitive), much faster than current methods. The potential of this technique demonstrated by determining resistance in both lab and clinical strains of E. coli, a key species for clinically burdensome urinary tract infections. This work provides the basis for a simple and fast diagnostic tool to detect antibiotic resistance in bacteria, reducing the health and economic burdens of AMR.

biophysics↗

bioRxiv2018

Single-molecule kinetics of pore assembly by the membrane attack complex

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins. C5b6 interacts with the lipid bilayer prior to recruiting C7 and C8. We discover that incorporation of the first C9 is the kinetic bottleneck of MAC formation, after which rapid C9 oligomerization completes the pore. This defines the kinetic basis for MAC assembly and provides insight into how human cells are protected from bystander damage by the cell surface receptor CD59, which is offered a maximum temporal window to halt the assembly at the point of C9 insertion.

biophysics↗