Single-molecule sequencing maps replication dynamics across the fission yeast genome, including centromeres

DNA replication dynamics in fission yeast remain incompletely understood, particularly in repetitive regions such as the centromeres and the rDNA. Here, we establish DNAscent—a nanopore sequencing method that detects BrdU-labelled nascent DNA and infers replication dynamics—to map replication at single-molecule resolution in fission yeast. For the first time, we have identified thousands of replication forks, as well as initiation, termination and pause events, on single sequenced molecules across the whole genome. This high coverage allowed us to identify replication patterns in poorly characterised regions. In the rDNA, we detect strand-specific pausing at replication fork barriers. At the mating-type locus, we find the most frequent pause site outside the rDNA. At centromeres, we find that replication initiation predominantly occurs in the outer repeats, while termination localises to central regions and that, only in centromere 2, there is an enrichment in pauses at the centromeric tRNAs. This work establishes a powerful single-molecule method for studying replication dynamics in fission yeast and provides insights into replication across repetitive regions that constitute a significant portion of the genomes of more complex organisms.