Replication dynamics of recombination-dependent replication forks

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.

Fig. 1. Polymerase usage following HR-restart.

a Schematic of the RTS1-rRFB locus on chromosome II. The positions of the directional RTS1 and rRFB barriers are shown as red and orange, respectively. The thick bar represents the directionality of fork arrest. ARS autonomously replicating sequence. The direction (see panel b) of unperturbed and perturbed replication at this locus is indicated by the thickness of arrows underneath. b Pu-seq traces of the ChrII locus. Top two traces: RTS1 barrier activity off (rtf1-d). Bottom two traces: RTS1 barrier activity on (rtf1⁺). Left: the usage of Pol δ (blue) and Pol ε (red) are shown on the Watson and Crick strands. Note the switch from Pol ε to Pol δ on the Watson strand at the RTS1 site is indicative of a change in polymerase usage on the leading strand when RTS1 barrier activity is on. Right: The same traces overlaid with data for Pol α (green). Pol α data is not to scale, see Methods section. c Chromatin immunoprecipitation of Rpa3-GFP at the indicated positions (relative to base 1 of the RTS1 sequence) in unsynchronised cells with either rnh1⁺ rnh201⁺ (WT) or rnh1-d rnh201-d backgrounds. Data presented are relative to the ade6⁺ control locus. n = 3 biological repeats. Error bars: standard deviation of the mean. d Chromatin immunoprecipitation using the S9.6 antibody in wild-type cells synchronised in G2 and released into S phase. n = 3 biological repeats. Error bars: standard deviation of the mean.

Fig. 2. HR-restarted replication does not mature from the δ/δ to ε/δ configuration.

a Schematic of the inverted RTS1 locus on chromosome II. The positions of the directional RTS1 barriers are shown in red, with the thick bar indicating the directionality of fork arrest. b Pu-seq traces from the modified locus when the barrier is active (adh-rtf1⁺). Note: the transition from Pol ε (red) to Pol δ (blue) on the Watson strand at the left-side RTS1 site and the transition from Pol ε to Pol δ on the Crick strand at right-side RTS1 site.

Fig. 3. HR-restarted forks are insensitive to the RTS1 barrier.

a Pu-seq traces for the RTS1-rRFB locus are overlaid for two sets of strains: rtf1⁺ (WT), which has endogenous rtf1 expression (Pol δ: blue, Pol: ε red) and adh-rtf1⁺, where expression is under control of the adh1 promoter (Pol δ: black, Pol: ε orange). b Schematic of the tandem RTS1-rRFB locus on chromosome II. The positions of the directional RTS1 and rRFB barriers are shown as red and orange, respectively with the thick bar indicating the directionality of fork arrest. ARS: autonomously replicating sequence. c Pu-seq traces for the tandem RTS1-rRFB locus which contains two copies of the RTS1 barrier that are both orientated to arrest left-right forks (right) compared to the original RTS1-rRFB locus (left). Top: barrier off. Bottom: barrier on.

Fig. 4. Loss of pku70⁺ results does not destabilise the leading stand primer.

a Overlays of Pu-seq traces of the RTS1-rRFB locus either with or without arrest and in the presence and absence of pku70⁺. Left: in the rnh201-d background. Right: in the rnh201-RED background. b For the purpose of comparison, overlays of the Pu-seq traces for rnh201-d and rnh201-RED backgrounds. Left: in pku70⁺. Right: in pku70-d.

Fig. 5. Schematic of HR-dependent restart.

(1) Canonical RF arrested at RTS1. DNA synthesised by Pol ε is shown in red. DNA synthesised by Pol δ shown in blue. Template: black. RTS1 sequence: orange. (2) Resection generates a free 3′ end. (3) Recombination factors generate a D-loop at the site of stalling. (4) The D-loop primer is extended, most likely by Pol δ. The D-loop is resolved, establishing semi-conservative DNA synthesis. (5). Pol δ, with a minimal contribution from Pol α, synthesises the leading strand. DNA synthesis is insensitive to further downstream RTS1 barriers. (6) Termination with the incoming canonical fork. (7). The gap is filled by Pol δ.