Single-molecule landscape of DNA replication pausing

Summary A pause in DNA synthesis that occurs when the replisome encounters an obstacle could lead to genome instability. Although important, systematic identification of replication pause sites is challenging due to their low frequency and delocalized nature. Here we present the first single-molecule identification of sites of replisome perturbation across a eukaryotic genome using long-read nanopore sequencing. For each single-molecule replication pause we determine the direction of replication, leading/lagging-strand identity, location and approximate duration, and whether the replisome resumed synthesis. Although pauses are largely diffuse over the genome, they are significantly enriched over transcribed features and correlate with transcription and R-loop levels. Transcription-replication conflicts are more numerous when head-on than co-directional. Finally, we identified genomic loci with a strong bias towards leading over lagging strand pauses, consistent with uncoupling of the helicase from polymerase epsilon. Our data support helicase-polymerase uncoupling resulting from replication pausing as the molecular trigger behind epigenetic switching.