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

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.