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

• Published in: Nature communications Vol. 16; no. 1; pp. 242 - 13
• Main Authors: Theulot, Bertrand, Tourancheau, Alan, Simonin Chavignier, Emma, Jean, Etienne, Arbona, Jean-Michel, Audit, Benjamin, Hyrien, Olivier, Lacroix, Laurent, Le Tallec, Benoît
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 02.01.2025; Nature Publishing Group; Nature Portfolio
• Subjects: 13/31; 13/44; 45/22; 45/23; 631/208/191; 631/337/151; 631/61/350/1058; Bromodeoxyuridine; Bromodeoxyuridine - metabolism; Deoxyribonucleic acid; DNA; DNA biosynthesis; DNA fingerprinting; DNA Replication; DNA Replication Timing; DNA sequencing; DNA, Fungal - genetics; DNA, Fungal - metabolism; Gene sequencing; High resolution; Humanities and Social Sciences; Life Sciences; multidisciplinary; Nanopore Sequencing - methods; Replication; Repressor Proteins; S phase; S Phase - genetics; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Science; Science (multidisciplinary); Single Molecule Imaging - methods; Synchronism; Telomere - genetics; Telomere - metabolism; Telomere-Binding Proteins - genetics; Telomere-Binding Proteins - metabolism; Telomeres; Yeast
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-55520-3

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. Current temporal studies of DNA replication are either complex or low-resolution. Here, the authors introduce Nanotiming, a simple and inexpensive method producing high-resolution, telomere-to-telomere replication timing profiles through BrdU-mediated capture of dTTP pool expansion in S phase.