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Bertrand Theulot1,2,3, Alan Tourancheau1, Emma Simonin Chavignier1

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Nanotiming, a new nanopore sequencing method, provides high-resolution DNA replication timing (RT) profiles. This technique reveals how Rif1 protein selectively delays replication of telomeres linked to specific subtelomeric elements.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Epigenetics

Background:

  • Current DNA replication timing (RT) studies lack resolution or require complex cell synchronization.
  • Understanding genome-wide RT is crucial for deciphering cellular processes and disease mechanisms.

Purpose of the Study:

  • To introduce Nanotiming, a novel single-molecule nanopore sequencing method for high-resolution, telomere-to-telomere RT profiling.
  • To analyze the role of Rif1 in regulating telomere replication timing.

Main Methods:

  • Nanotiming interrogates intracellular dTTP concentration changes during S phase by competing dTTP with bromodeoxyuridine triphosphate (BrdUTP) for DNA incorporation.
  • Asynchronously growing cells are labeled with BrdU, and its incorporation is quantified along nanopore sequencing reads.
  • The method was validated in *S. cerevisiae*, comparing Nanotiming RT profiles with established methods in wild-type and mutant cells.

Main Results:

  • Nanotiming successfully reproduced established RT profiles in *S. cerevisiae*, demonstrating its accuracy.
  • The method achieved high resolution, enabling telomere-to-telomere RT analysis.
  • Nanotiming revealed that the telomere regulator Rif1 selectively delays the replication of telomeres associated with specific subtelomeric elements.

Conclusions:

  • Nanotiming is a simple, accurate, and cost-effective method for high-resolution genome-wide RT analysis.
  • The technique offers unique capabilities for studying individual telomere replication timing.
  • Rif1's role in regulating telomere replication timing based on subtelomeric elements was elucidated.