Biology subjects

Wilkinson, E. M.

Publications and source records attributed to Wilkinson, E. M..

3 recordsLinked to original sources

bioRxiv2026

Direct visualisation of post-replication gap formation at the bacterial RRS

Replication risk sequences (RRS) are recently discovered genomic structural elements that trigger post-replication gap formation during replisome passage. In E. coli, the two RRS elements are nearly perfect 222-bp G-quadruplex-containing repeats that flank the terminal domain and are highly conserved in both sequence and genomic position across enterobacteria. We report here the first direct visualisation of RRS function in vitro using single-molecule methods. When the G4 strand of the RRS element is positioned on the lagging-strand template, gaps are formed essentially every time a replisome encounters it. An increase in ssDNA in the synthesised DNA is readily seen using ssGAP-seq methods. When the G-quadruplex strand of the RRS is positioned on the leading-strand template, gaps are formed, albeit at lower frequency. However, the continued DNA synthesis in a rolling-circle assay indicates that the gaps are still formed on the lagging strand, indicating that the RRS complementary strand has a significant but reduced capacity to form a structure that triggers lesion skipping. The results document the potency of the RRS as a trigger for gap formation, suggesting a possible function for at least some eukaryotic G-quadruplexes.

biophysics↗

bioRxiv2025

The interplay between supercoiling and DNA modifying enzymes at the single-molecule level

DNA supercoiling refers to the overwinding or underwinding of the double helix, forming plait-like structures called plectonemes. Supercoiling is involved in all biological processes that occur on DNA. Despite this involvement, single-molecule in vitro studies on these processes largely work with relaxed DNA, overlooking the role supercoiling is playing in these processes. We have constructed 18-kb linear DNA substrates that can be tethered to a microscope coverslip at multiple sites, allowing the template to be topologically constrained. We can then use an intercalating dye to induce positive or negative supercoiling, which we can observe at the single-molecule level using TIRF microscopy. We directly visualise plectonemes diffusing along the DNA. However, in the presence of the restriction enzyme EcoRV, plectonemes remain stationary at the EcoRV binding site. We show that EcoRV has an increased cleavage efficiency in the presence of supercoiling, even though its binding efficiency is unaffected. These findings reveal a relationship between DNA topology and the activity of DNA modifying enzymes. We hypothesise that supercoiling makes cleavage more energetically favourable for restriction enzymes that bend DNA. This work reveals the kinetic interplay between supercoiling and enzyme activity, highlighting how DNA itself can modulate the processes that act upon it. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/680164v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1a9890forg.highwire.dtl.DTLVardef@1653ecaorg.highwire.dtl.DTLVardef@1fd7bfcorg.highwire.dtl.DTLVardef@d70357_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Effects of neural noise on predictive model updating across the adult lifespan

AbstractIn the perceptual and sensorimotor domains, ageing is accompanied by a stronger reliance on top-down predictive model information and reduced sensory learning, thus promoting simpler, more efficient internal models in older adults. Here, we demonstrate analogous effects in higher-order language processing. One-hundred and twenty adults ranging in age from 18 to 83 years listened to short auditory passages containing manipulations of adjective order, with order probabilities varying between two speakers. As a measure of model adaptation, we examined attunement of the N400 event-related potential, a measure of precision-weighted prediction errors in language, to a trial-by-trial measure of speaker-based adjective order expectedness ("speaker-based surprisal") across the course of the experiment. Adaptation was strongest for young adults, weaker for middle-aged adults, and absent for older adults. Over and above age-related differences, we observed individual differences in model adaptation, with aperiodic (1/f) slope and intercept metrics derived from resting-state EEG showing the most pronounced modulations. We suggest that age-related changes in aperiodic slope, which have been linked to neural noise, may be associated with individual differences in the magnitude of stimulus-related prediction error signals. By contrast, changes in aperiodic intercept, which reflects aggregate population spiking, may relate to an individuals updating of inferences regarding stimulus precision. These two mechanisms jointly contribute to age-related changes in the precision-weighting of prediction errors and the degree of sensory learning.

neuroscience↗