Biology subjects

Pfuderer, P. L.

Publications and source records attributed to Pfuderer, P. L..

4 recordsLinked to original sources

bioRxiv2026

A genome language model for mapping DNA replication origins

Origin firing is a central process during DNA replication, but specific sequences defining replication origin usage have not been defined in human cells. Here, we show that a genome language model can accurately predict which sequences can act as an origin of replication, thereby enabling the fast and cost-effective creation of genome-wide replication origin maps. We fine-tuned a genome language model on the primary sequence of mapped human origins to establish ORILINX (ORIgin of replication Language-model Inference via Nucleotide conteXt) and found that it learns a rich representation of sequence features linked to replication initiation, extending beyond known predictive features such as GC-content and G-quadruplex motifs. When applied genome-wide, the models sequence-derived origin calling closely mirrors origin efficiency inferred from replication timing, suggesting that intrinsic sequence context encodes information relevant to initiation frequency. Furthermore, we performed Short Nascent Strand sequencing (SNS-seq) and Repli-seq to demonstrate that ORILINX can generalise to other mammalian genomes, such as those of mice and sheep, as well as other vertebrates such as chickens. Finally, we packaged ORILINX into a simple, easy-to-use tool which is available at https://github.com/Pfuderer/ORILINX.git.

genomics↗

bioRxiv2025

ecDNA replication is disorganised and vulnerable to replication stress

Extrachromosomal DNA (ecDNA) is a critical driver of cancer progression, contributing to tumour growth, evolution, and therapeutic resistance through oncogene amplification. Despite its significance, the replication of ecDNA remains poorly understood. In this study, we investigated the replication dynamics of ecDNA using high-resolution replication timing analysis (Repli-seq) and DNAscent, a method for measuring origin firing and replication fork movement based on ultra-long read Oxford Nanopore Sequencing, that we applied to both bulk DNA and to ecDNA isolated with FACS-based Isolation of Native ecDNA (FINE), a new method for isolating intact, chromatinised ecDNA without DNA or protein digestion. We demonstrate that ecDNA in the COLO 320DM colorectal cancer cell line exhibits largely asynchronous replication throughout the S phase, contrasting with the conserved replication timing of the corresponding normal linear chromosomal DNA in RPE-1 cells and the chromosomally reintegrated ecDNA in COLO 320HSR, which forms a homogeneously staining region. Replication origins on ecDNA are redistributed, and replication forks exhibit reduced velocity and increased stalling, particularly near the c-MYC oncogene. Under replication stress induced by hydroxyurea treatment, ecDNA replication is further compromised, leading to altered origin activation, reduced fork velocity and eventual ecDNA depletion from cells. Our findings reveal fundamental differences in the replication dynamics of ecDNA, providing insights that could inform the development of therapies targeting ecDNA-associated oncogene amplification in cancer.

cancer biology↗

bioRxiv2025

High levels of DNA replication initiation factors indicate ATR inhibitor sensitivity via excessive origin firing

Inhibitors of ATR, a central kinase controlling DNA replication origin firing and cellular checkpoint activity, are currently in multiple clinical trials, yet mechanisms underpinning sensitivity and robust patient stratification biomarkers are lacking. We used functional genomics approaches to identify molecular mechanisms driving sensitivity to the ATR inhibitor (ATRi) ceralasertib. Replication stress-associated patterns of DNA copy number alterations identified a subset of sensitive breast cancer cell lines. In parallel, we performed proteomics, phosphoproteomics and gene expression analyses and discovered that sensitive cell lines had higher expression of DNA replication origin firing factors, and massively increased origin firing in response to ATRi. ATRi sensitivity was partly rescued upon co-treatment with XL-413, a CDC7 inhibitor that decreases origin firing. High expression of replication initiation factors correlated with ATRi sensitivity across multiple cancer types, and in acute myeloid leukemia patient samples. Together, this study reveals a novel contribution of lethal origin firing capacity in determining the sensitivity of cancer cells to ATR inhibition and demonstrates the predictive potential of mechanism-specific copy number alterations, providing key steps towards developing a multimodal clinically applicable biomarker for ATR inhibitors.

cancer biology↗

bioRxiv2022

A high-resolution, nanopore-based artificial intelligence assay for DNA replication stress in human cancer cells

DNA replication stress is a hallmark of cancer that is exploited by chemotherapies. Current assays for replication stress have low throughput and poor resolution whilst being unable to map the movement of replication forks genome-wide. We present a new method that uses nanopore sequencing and artificial intelligence to map forks and measure their rates of movement and stalling in melanoma and colon cancer cells treated with chemotherapies. Our method can differentiate between fork slowing and fork stalling in cells treated with hydroxyurea, as well as inhibitors of ATR, WEE1, and PARP1. These different therapies yield different characteristic signatures of replication stress. We assess the role of the intra-S-phase checkpoint on fork slowing and stalling and show that replication stress dynamically changes over S-phase. This method requires sequencing on only a single nanopore flow cell, and the cost-effectiveness and high throughput enables functional screens to determine how human cancers respond to replication-targeted therapies.

cancer biology↗