Single-molecule mapping of replisome progression

Abstract

AbstractFundamental 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 new 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 molecules. Using Replicon-seq, we have investigated replisome movement along chromatin. We found that sister replisomes progress with remarkable consistency from the origin of replication but function autonomously. 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 facilitating efficient termination. Replicon-seq provides an unprecedented means of defining replisome movement across the genome.