Biology subjects

Rogers, A. M.

Publications and source records attributed to Rogers, A. M..

3 recordsLinked to original sources

bioRxiv2025

Single-molecule landscape of DNA replication pausing

A pause in DNA synthesis that occurs when the replisome encounters an obstacle could lead to genome instability. Although important, systematic identification of replication pause sites is challenging due to their low frequency and delocalized nature. Here we present the first single-molecule identification of sites of replisome perturbation across a eukaryotic genome using long-read nanopore sequencing. For each single-molecule replication pause we determine the direction of replication, leading/lagging-strand identity, location and approximate duration, and whether the replisome resumed synthesis. Although pauses are largely diffuse over the genome, they are significantly enriched over transcribed features and correlate with transcription and R-loop levels. Transcription-replication conflicts are more numerous when head-on than co-directional. Finally, we identified genomic loci with a strong bias towards leading over lagging strand pauses, consistent with uncoupling of the helicase from polymerase epsilon. Our data support helicase-polymerase uncoupling resulting from replication pausing as the molecular trigger behind epigenetic switching. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/670160v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@79250forg.highwire.dtl.DTLVardef@1702a30org.highwire.dtl.DTLVardef@e1d9f4org.highwire.dtl.DTLVardef@1ac2ce1_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Single-molecule sequencing maps replication dynamics across the fission yeast genome, including centromeres

DNA replication dynamics in fission yeast remain incompletely understood, particularly in repetitive regions such as the centromeres and the rDNA. Here, we establish DNAscent--a nanopore sequencing method that detects BrdU-labelled nascent DNA and infers replication dynamics--to map replication at single-molecule resolution in fission yeast. For the first time, we have identified thousands of replication forks, as well as initiation, termination and pause events, on single sequenced molecules across the whole genome. This high coverage allowed us to identify replication patterns in poorly characterised regions. In the rDNA, we detect strand-specific pausing at replication fork barriers. At the mating-type locus, we find the most frequent pause site outside the rDNA. At centromeres, we find that replication initiation predominantly occurs in the outer repeats, while termination localises to central regions and that, only in centromere 2, there is an enrichment in pauses at the centromeric tRNAs. This work establishes a powerful single-molecule method for studying replication dynamics in fission yeast and provides insights into replication across repetitive regions that constitute a significant portion of the genomes of more complex organisms.

genomics↗

bioRxiv2023

A 3D in vitro cortical tissue model based on dense collagen to study the effects of gamma radiation on neuronal function

Studies on gamma radiation-induced injury have long been focused on hematopoietic, gastrointestinal, and cardiovascular systems, yet little is known about the effects of gamma radiation on the function of human cortical tissue. The challenge in studying radiation-induced cortical injury is, in part, due to a lack of human tissue models and physiologically relevant readouts. Here, we have developed a physiologically relevant 3D collagen-based cortical tissue model (CTM) for studying the functional response of human iPSC derived neurons and astrocytes to a sub-lethal radiation exposure (5 Gy). We quantified cytotoxicity, DNA damage, morphology, and extracellular electrophysiology. We reported that 5 Gy exposure significantly increased cytotoxicity, DNA damage, and astrocyte reactivity while significantly decreased neurite length and neuronal network activity. Additionally, we found that clinically deployed radioprotectant amifostine ameliorated the DNA damage, cytotoxicity, and astrocyte reactivity. The CTM provides a critical experimental platform to understand cell-level mechanisms by which GR affects human cortical tissue and to screen prospective radioprotectant compounds.

bioengineering↗