Replication dynamics of recombination-dependent replication forks

Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Here, we use polymerase usage sequencing to visualize in vivo replication dynamics of HR-restarted forks at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation. We show that HR-restarted forks synthesise both strands with Pol δ for up to 30 kb without maturing to a δ/ε configuration and that Pol α is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol δ later. We further demonstrate that HR-restarted forks progress uninterrupted through a fork barrier that arrests canonical forks. Finally, by manipulating lagging strand resection during HR-restart by deleting pku70, we show that the leading strand initiates replication at the same position, signifying the stability of the 3′ single strand in the context of increased resection.