Abstract
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.
Highlights
Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ
Replication forks are efficiently arrested at RTS1, a programmed unidirectional replication fork barrier of ~850 bp DNA derived from the mating type locus of fission yeast[20]
The distant (~30 kb away) late origin is activated, but the difference in timing of origin activation and the time taken for fork movement means that the converging right-to-left moving fork does not reach the RTS1 sequence and the homologous recombination (HR)-restarted Replication forks (RFs) travels left-to-right for ~5–15 kb, until it terminates with the convergent right-left fork
Summary
Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Restart can occur from a one-ended DNA double strand break (DSB), such as would occur if the RF encounters a nick[8], or from a resected replication fork, likely following fork reversal that generates a 4-way DNA junction known as a chicken-foot[9] To explore the latter mechanism of HR-restart of collapsed RFs we developed a model system in fission yeast using a unidirectional replication fork barrier, RTS1. Using this system, we and others have demonstrated that HR-dependent restart occurs rapidly (~18 min10), does not require a DSB intermediate[11] and likely involves an initial fork reversal step that allows regulation of resection via Ku binding and subsequent displacement by the Mre11-Rad50-Nbs[1] complex[12]. We found that, unlike canonical replication (where polymerase Pol ε synthesises the leading strand and Pol δ the lagging strand), both the leading and lagging strands are synthesised by Pol δ despite the fact that replication remains semi-conservative[10] and does not progress as a migrating D-loop as is seen during break-induced replication[16]
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