Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming

Bertrand Theulot, Alan Tourancheau, Emma Simonin Chavignier, Etienne Jean, Jean-Michel Arbona, Benjamin Audit, Olivier Hyrien, Laurent Lacroix and Benoît Tallec ()
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Bertrand Theulot: École normale supérieure, Université PSL, CNRS, INSERM
Alan Tourancheau: École normale supérieure, Université PSL, CNRS, INSERM
Emma Simonin Chavignier: École normale supérieure, Université PSL, CNRS, INSERM
Etienne Jean: École normale supérieure, Université PSL, CNRS, INSERM
Jean-Michel Arbona: École Normale Supérieure de Lyon, CNRS, UMR5239, INSERM, U1293, Université Claude Bernard Lyon 1
Benjamin Audit: LPENSL, UMR5672
Olivier Hyrien: École normale supérieure, Université PSL, CNRS, INSERM
Laurent Lacroix: École normale supérieure, Université PSL, CNRS, INSERM
Benoît Tallec: École normale supérieure, Université PSL, CNRS, INSERM

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: Abstract Current temporal studies of DNA replication are either low-resolution or require complex cell synchronisation and/or sorting procedures. Here we introduce Nanotiming, a single-molecule, nanopore sequencing-based method producing high-resolution, telomere-to-telomere replication timing (RT) profiles of eukaryotic genomes by interrogating changes in intracellular dTTP concentration during S phase through competition with its analogue bromodeoxyuridine triphosphate (BrdUTP) for incorporation into replicating DNA. This solely demands the labelling of asynchronously growing cells with an innocuous dose of BrdU during one doubling time followed by BrdU quantification along nanopore reads. We demonstrate in S. cerevisiae model eukaryote that Nanotiming reproduces RT profiles generated by reference methods both in wild-type and mutant cells inactivated for known RT determinants. Nanotiming is simple, accurate, inexpensive, amenable to large-scale analyses, and has the unique ability to access RT of individual telomeres, revealing that Rif1 iconic telomere regulator selectively delays replication of telomeres associated with specific subtelomeric elements.

Date: 2025
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DOI: 10.1038/s41467-024-55520-3

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