Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming
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)...
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| Format: | Article |
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55520-3 |
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| author | Bertrand Theulot Alan Tourancheau Emma Simonin Chavignier Etienne Jean Jean-Michel Arbona Benjamin Audit Olivier Hyrien Laurent Lacroix Benoît Le Tallec |
| author_facet | Bertrand Theulot Alan Tourancheau Emma Simonin Chavignier Etienne Jean Jean-Michel Arbona Benjamin Audit Olivier Hyrien Laurent Lacroix Benoît Le Tallec |
| author_sort | Bertrand Theulot |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-201a262882814e3890c5915e16b9cee8 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
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| series | Nature Communications |
| spelling | doaj-art-201a262882814e3890c5915e16b9cee82025-01-05T12:37:30ZengNature PortfolioNature Communications2041-17232025-01-0116111310.1038/s41467-024-55520-3Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with NanotimingBertrand Theulot0Alan Tourancheau1Emma Simonin Chavignier2Etienne Jean3Jean-Michel Arbona4Benjamin Audit5Olivier Hyrien6Laurent Lacroix7Benoît Le Tallec8IBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMIBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMIBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMIBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMLaboratoire de Biologie et Modélisation de la Cellule, École Normale Supérieure de Lyon, CNRS, UMR5239, INSERM, U1293, Université Claude Bernard Lyon 1CNRS, ENS de Lyon, LPENSL, UMR5672IBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMIBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMIBENS, Département de biologie, École normale supérieure, Université PSL, CNRS, INSERMAbstract 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.https://doi.org/10.1038/s41467-024-55520-3 |
| spellingShingle | Bertrand Theulot Alan Tourancheau Emma Simonin Chavignier Etienne Jean Jean-Michel Arbona Benjamin Audit Olivier Hyrien Laurent Lacroix Benoît Le Tallec Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming Nature Communications |
| title | Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming |
| title_full | Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming |
| title_fullStr | Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming |
| title_full_unstemmed | Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming |
| title_short | Telomere-to-telomere DNA replication timing profiling using single-molecule sequencing with Nanotiming |
| title_sort | telomere to telomere dna replication timing profiling using single molecule sequencing with nanotiming |
| url | https://doi.org/10.1038/s41467-024-55520-3 |
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