Replication dynamics of recombination-dependent replication forks

Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol delta. 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 delta for up to 30kb without maturing to a delta/epsilon configuration and that Pol alpha is not used significantly on either strand, suggesting the lagging strand template remains as a gap that is filled in by Pol delta 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. Replication forks that are stalled at obstacles on the DNA template can be restarted by homologous recombination. Here, the authors show replication dynamics during homologous recombination-dependent replication fork restart by combining polymerase usage sequencing and a Monte Carlo mathematical model.

Automated summary

Replication forks restarted by homologous recombination utilize Pol delta to synthesize both strands without maturing to a delta/epsilon configuration, while the template for the lagging strand remains as a gap filled in by Pol delta later. These restarted forks are able to progress through barriers that arrest canonical forks, demonstrating stability of the 3' single strand during increased resection.