Single-molecule mapping of replisome progression

Fundamental aspects of DNA replication, such as the anatomy of replication stall sites, how replisomes are influenced by gene transcription, and whether the progression of sister replisomes is coordinated, are poorly understood. Available techniques do not allow the precise mapping of the positions of individual replisomes on chromatin. We have developed a method called Replicon-seq that entails the excision of full-length replicons by controlled nuclease cleavage at replication forks. Replicons are sequenced using Nanopore, which provides a single-molecule readout of long DNA. Using Replicon-seq, we found that sister replisomes function autonomously and yet progress through chromatin with remarkable consistency. Replication forks that encounter obstacles pause for a short duration but rapidly resume synthesis. The helicase Rrm3 plays a critical role both in mitigating the effect of protein barriers and with facilitating efficient termination. Replicon-seq provides a high-resolution means of defining how individual replisomes move across the genome.

Automated summary

This study developed a method called Replicon-seq, which can accurately define the movement of individual replisomes across the genome. Using this method, the study found that sister replisomes function autonomously and progress through chromatin with remarkable consistency. Replication forks pause briefly when encountering obstacles but quickly resume synthesis, and the helicase Rrm3 plays a critical role in mitigating the effect of protein barriers and facilitating efficient termination.