Genome-wide mapping of individual replication fork velocities using nanopore sequencing

Area of Science:

• Genomics
• Molecular Biology
• Biotechnology

Background:

• Determining eukaryotic DNA replication fork velocity is challenging due to limitations in existing techniques.
• Current methods often lack sequence information or have low throughput, hindering genome-wide analysis.

Purpose of the Study:

• To introduce NanoForkSpeed, a novel nanopore sequencing-based method for mapping and quantifying individual DNA replication fork velocities.
• To provide a high-throughput, sequence-informed approach to study replication dynamics in eukaryotic genomes.

Main Methods:

• Utilized nanopore sequencing to analyze tracks of bromodeoxyuridine incorporated during pulse-labeling of cells.
• Developed NanoForkSpeed to detect and extract velocities of individual replication forks from sequencing data.
• Applied the method to Saccharomyces cerevisiae (yeast) to generate a genome-wide map of fork speeds.

Main Results:

• NanoForkSpeed successfully reproduced established mean replication fork speed estimates in yeast (≈2 kb/min).
• The method precisely quantified speed alterations in cells with modified replisome progression or treated with hydroxyurea.
• A genome-wide map of over 125,000 individual fork velocities revealed uniform fork speeds across yeast chromosomes, with notable slowdowns at known pausing sites.

Conclusions:

• NanoForkSpeed is an effective tool for high-throughput, individual replication fork velocity measurement in eukaryotes.
• The study provides a detailed genome-wide map of replication fork progression, highlighting conserved pausing sites.
• This method advances our understanding of DNA replication dynamics and its regulation.