Biology subjects

Willems, J.

Publications and source records attributed to Willems, J..

4 recordsLinked to original sources

bioRxiv2026

Mutagenic bypass of uracil-derived abasic sites underlies the SBS17 mutational signature

Understanding how mutations arise is central to predicting and preventing cancer, yet most of the mutational signatures found in cancer genomes still have no known mechanistic cause. Among these, single-base-substitution signature 17 (SBS17) is the dominant mutational process in gastric and oesophageal adenocarcinoma. SBS17 has been linked to oxidative damage and to the chemotherapeutic agent 5-fluorouracil (5-FU), but the molecular events that generate it are unknown. Here we demonstrate that SBS17 causes driver mutations in gastrointestinal cancer. We then combine genetics in cell lines and organoids with whole-genome sequencing to define the mechanistic aetiology of SBS17 mutagenesis. We disprove the prevailing oxidative damage hypothesis and instead show that SBS17 arises through the misincorporation of dUTP during DNA replication, followed by uracil excision by the glycosylase UNG and mutagenic bypass of the resulting abasic (AP) sites by the translesion synthesis machinery. Importantly, we reveal that the same mechanism underlies both spontaneous and chemotherapy-induced mutations. Together, our results resolve the origin of gastric mutagenesis, opening therapeutic avenues for curbing ongoing mutagenesis, tumour evolution and drug resistance in gastrointestinal cancers.

molecular biology↗

bioRxiv2025

Tissue-specific mutagenesis from endogenous guanine damageis suppressed by Polκ and DNA repair

Knowledge of mutational patterns has expanded significantly, but a persistent challenge is to link these complex patterns to the underlying molecular mechanism or source of DNA damage, especially when that damage is endogenous and not driven by environmental mutagens. Technological advances now allow us to catalogue mutation across tissues or even closely related cell types, but the results are largely descriptive until we identify the endogenous sources of mutation and how they differ across tissues. Here, we combine mouse genetics, advanced sequencing, biochemistry and mass spectrometry to provide a detailed mechanistic understanding of endogenous mutagenesis. We reveal that endogenous guanine adducts are significant drivers of tissue-specific mutagenesis, while the interwoven actions of DNA polymerase Pol{kappa} and DNA repair mechanisms are pivotal in mitigating mutagenesis. For the first time, we use untargeted DNA adductomics to characterize new sources of endogenous DNA damage. Our novel approach to understanding endogenous mutational landscapes reveals previously unseen mutational processes and points to vast potential for new discoveries.

molecular biology↗

bioRxiv2022

Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.

neuroscience↗

bioRxiv2019

ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

The correct subcellular distribution of protein complexes establishes the complex morphology of neurons and is fundamental to their functioning. Thus, determining the dynamic distribution of proteins is essential to understand neuronal processes. Fluorescence imaging, in particular super-resolution microscopy, has become invaluable to investigate subcellular protein distribution. However, these approaches suffer from the limited ability to efficiently and reliably label endogenous proteins. We developed ORANGE: an Open Resource for the Application of Neuronal Genome Editing, that mediates targeted genomic integration of fluorescent tags in neurons. This toolbox includes a knock-in library for in-depth investigation of endogenous protein distribution, and a detailed protocol explaining how knock-in can be developed for novel targets. In combination with super-resolution microscopy, ORANGE revealed the dynamic nanoscale organization of endogenous neuronal signaling molecules, synaptic scaffolding proteins, and neurotransmitter receptors. Thus, ORANGE enables quantitation of expression and distribution for virtually any protein in neurons at high resolution and will significantly further our understanding of neuronal cell biology.

neuroscience↗