Biology subjects

Boulton, S.

Publications and source records attributed to Boulton, S..

4 recordsLinked to original sources

bioRxiv2026

Phenotype-first covalent fragment screening identifies a synthetic lethal TYMS inhibitor in ATRX-deficient cells

Chemoproteomic mapping of covalent fragment libraries is expanding the ligandable human proteome with direct evidence of cellular target engagement. However, understanding the functional consequences of specific covalent modifications typically requires extensive downstream biological characterisation. Here we present a phenotype-first approach that integrates covalent fragment screening with chemoproteomics and genetic deconvolution in a disease-relevant context. Using isogenic ATRX wild-type and knockout eHAP iCAS9 cells, we screened a library of around 500 cysteine-reactive fragments for differential cell killing and identified a chloroacetamide fragment, PP12, that selectively impairs the viability of ATRX-deficient cells. By combining competitive click-chemoproteomics with genome-wide CRISPR synthetic lethal datasets, we identified thymidylate synthase (TYMS) as a phenotypically relevant target of PP12. Target validation was supported by crystallography, competition with the active-site inhibitor 5-fluorouracil, and impaired dTMP synthesis in cells. Mechanistically, TYMS inhibition induces replication stress that is selectively cytotoxic to ATRX-deficient cells and is dependent on FAM111A and SLFN11. This work establishes a generalisable workflow linking covalent fragment phenotypes to target deconvolution using chemoproteomics and mechanistic validation.

cell biology↗

bioRxiv2022

RTEL-1 and DNA polymerase theta promote subtelomeric DNA synthesis and telomere fusion in C. elegans

Interstitial telomere sequences (ITS) are degenerate telomere tracts scattered along chromosome arms whose functions are not well understood. We found that critically shortened telomeres of C. elegans telomerase mutants initiate DNA synthesis within ITS tracts that were close to or far from a telomere. Some ITS tracts were targeted recurrently. RTEL-1 dismantles T-loops and recombination intermediates, and DNA polymerase theta (POLQ-1) promotes end-joining using short segments of microhomology. In telomerase mutants, RTEL-1 and POLQ-1 promoted telomere fusion and DNA synthesis at subtelomeric ITS tracts. RTEL-1 is known to suppress homologous recombination, and we found that RTEL-1 similarly suppressed POLQ-1-mediated double-strand break repair. Mutation signatures characteristic of repair by POLQ-1 occurred during initiation of subtelomeric DNA synthesis and at subsequent template shifting events. We propose that RTEL-1 and POLQ-1 play distinct essential roles in subtelomeric DNA synthesis, a process that may contribute significantly to telomere fusion and tumor genome evolution.

genetics↗

bioRxiv2021

Characterization of Critical Determinants of ACE2-RBD Interaction

Despite sequence similarity to SARS-CoV-1, SARS-CoV-2 has demonstrated greater widespread virulence and unique challenges to researchers aiming to study its pathogenicity in humans. The interaction of the viral receptor binding domain (RBD) with its main host cell receptor, angiotensin-converting enzyme 2 (ACE2), has emerged as a critical focal point for the development of anti-viral therapeutics and vaccines. Utilizing our recently developed NanoBiT technology-based biosensor, we selectively identify and characterize the impact of mutating certain amino acid residues in the RBD of SARS-CoV-2 and in ACE2. Specifically, we examine the mutational effects on RBD-ACE2 binding ability, before and after the addition of competitive inhibitors, as well as neutralizing antibody activity. These critical determinants of virus-host interactions may provide more effective targets for ongoing vaccines, drug development, and potentially pave the way for determining the genetic variation underlying disease severity.

microbiology↗

bioRxiv2020

Systematic analysis of mutational spectra associated with DNA repair deficiency in C. elegans

Genome integrity is particularly important in germ cells to faithfully preserve genetic information across generations. As yet little is known about the contribution of various DNA repair pathways to prevent mutagenesis. Using the C. elegans model we analyse mutational spectra that arise in wild-type and 61 DNA repair and DNA damage response mutants cultivated over multiple generations. Overall, 44% of lines show >2-fold increased mutagenesis with a broad spectrum of mutational outcomes including changes in single or multiple types of base substitutions induced by defects in base excision or nucleotide excision repair, or elevated levels of 50-400 bp deletions in translesion polymerase mutants rev-3(pol {zeta}) and polh-1(pol {eta}). Mutational signatures associated with defective homologous recombination fall into two classes: 1) mutants lacking brc-1/BRCA1 or rad-51/RAD51 paralogs show elevated base substitutions, indels and structural variants, while 2) deficiency for MUS-81/MUS81 and SLX-1/SLX1 nucleases, and HIM-6/BLM, HELQ-1/HELQ and RTEL-1/RTEL1 helicases primarily cause structural variants. Genome-wide investigation of mutagenesis patterns identified elevated rates of tandem duplications often associated with inverted repeats in helq-1 mutants, and a unique pattern of translocation events involving homeologous sequences in rip-1 paralog mutants. atm-1/ATM DNA damage checkpoint mutants harboured complex structural variants enriched in subtelomeric regions, and chromosome end-to-end fusions. Finally, while inactivation of the p53-like gene cep-1 did not affect mutagenesis, combined brc-1 cep-1 deficiency displayed increased, locally clustered mutagenesis. In summary, we provide a global view of how DNA repair pathways prevent germ cell mutagenesis.

genomics↗