Biology subjects

Holmes, E. P.

Publications and source records attributed to Holmes, E. P..

3 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↗

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↗