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. 2015 Nov;22(11):932-8.
doi: 10.1038/nsmb.3100. Epub 2015 Oct 5.

Polymerase δ replicates both strands after homologous recombination-dependent fork restart

Izumi Miyabe  1 , Ken'Ichi Mizuno  1 , Andrea Keszthelyi  1 , Yasukazu Daigaku  2 , Meliti Skouteri  1 , Saed Mohebi  1 , Thomas A Kunkel  3 , Johanne M Murray  1 , Antony M Carr  1
Affiliations

Polymerase δ replicates both strands after homologous recombination-dependent fork restart

Izumi Miyabe et al. Nat Struct Mol Biol. 2015 Nov.

Abstract

To maintain genetic stability, DNA must be replicated only once per cell cycle, and replication must be completed even when individual replication forks are inactivated. Because fork inactivation is common, passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via homologous recombination-restarted replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis because HoRReR is more error prone than canonical replication. This increased error rate implies the HoRReR mechanism is distinct from that of a canonical fork. Here we demonstrate, in Schizosaccharomyces pombe, that a DNA sequence duplicated by HoRReR during S phase is replicated semiconservatively, but both the leading and lagging strands are synthesized by DNA polymerase δ.

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Figures

Figure 1
Figure 1. Polδ, but not Polε participates in bulk DNA synthesis after HR-restart
(a) Schematic representation of the construct containing RTS1 and repetitive TER2/3 (T45R). The brown and black region is always replicated by a canonical fork. The red and blue region is replicated canonically when barrier activity is OFF, while it is replicated by Homologous Recombination Restarted Replication (HoRReR) when the barrier activity is active ON. (b) Delayed timing of DNA synthesis after HR-restart. Genomic DNA was extracted at the indicated time points from G2 arrested cdc25-22 cells released synchronously into the cell cycle. Replication of the indicated loci, U5 and L3, was monitored by quantitative PCR. (c) The contribution of Polε to DNA synthesis during HoRReR. (d) Equivalent analysis of Polδ contribution to DNA synthesis during HoRReR.
Figure 2
Figure 2. Polδ usage is relatively uniform and Polα usage decreased
(a) Polδ extends a minimum of several kb of the leading strand during homologous recombination restarted replication. The ura5 (ClaI-BlpI) and ura4 (HindIII-ClaI) regions were analysed separately. Genomic DNA from the strains indicated was digested, alkali treated and separated on an alkaline gel. The Watson and Crick strands were visualized following alkali treatment using single-stranded probes as indicated by colors of frames (c.f. Fig. 1a). Non-specific band; *. (b) Spontaneous mutation rates for indicated strains in a swi7-H4 (Polα) background. ON and OFF represent rtf1+ and rtf1-d, respectively. Values: mean of three independent experiments, each with 11 independent cultures. Error bars: s.d.
Figure 3
Figure 3. HR-restarted replication is semi-conservative
(a) Possible models for progression of Homologous Recombination Restarted Replication (HoRReR). Top: following strand invasion, replication occurs via a migrating D-loop. The newly synthesised strand is used as a template for the “lagging” strand, resulting in conservative replication. Bottom: Following strand invasion and D-loop formation, the replication fork is reset such that both parental strands template new synthesis, resulting in semi-conservative replication. (b) Schematic representation of DNA fragment analysed. (c) Density substitution analysis of HR-restarted replication. Sample are digested DNA from rnh201-d polδ-L591G cells at (T0) or after 150 min incubation in a heavy medium (T150). The distribution of the ura4-ura5 fragment after CsCl gradient centrifugation is shown. Upper panel: rtf1+ (RTS1 barrier activity ON). HH+LL indicates distribution of control DNA (mixture of heavy:heavy and light:light plasmid DNA). Lower panel: rtf1-d (RTS1 barrier activity OFF). Error bars: s.d. n=3. (d) For density substitution experiments, the leading strand is synthesised by Polδ after HoRReR (see Fig. 1d). The leading and lagging strands of ura4-ura5 were detected by single-stranded probes. Density substitution experiments in a cdc6+ (Polδ+) rnh2+ background (where HoRReR cannot be verified at the time of the experiment) show equivalent results.
Figure 4
Figure 4. Instability resulting from homologous recombination restarted replication is not intrinsic to leading strand synthesis by Polδ
(a) Both leading and lagging stands are synthesised by Polδ in the Polε N-terminal deletion strain. Alkali sensitivity in the indicated strains was assessed by Southern blot using strand-specific probes. (b) Schematic of the Rura4-sd20 locus for the microhomology mediated strand exchange assay. ura4-sd20 contains a 20bp duplication flanked by microhomology and is phenotypically ura− (c) Replication slippage at Rura4-sd20 measured in a cdc20+ (Polε+) and a cdc20-ΔN strain where the catalytic domain of Polε is deleted. Replication forks were either not arrested (OFF: rtf1Δ) or arrested (ON: rtf1+) at RTS1. Error bars; s.d. n=3, each with 11 independent cultures. (d) Schematic of the TpalR locus used for the Gross Chromosomal Rearrangement (GCR) assay. Homologous recombination restarted replication results in fork U-turn at the inverted repeat center, causing acentric and dicentric chromosome formation. (e) Southern blot of normal (9.9 kb) and dicentric (14.4 kb) chromosomes in three independent isolates of TpalR cdc20+ (Polε+) and TpalR cdc20-ΔN in an rtf1Δ background (no arrest). A low level of uncharacterized higher molecular weight species is evident in cdc20-ΔN cells (vertical bar: ?). Right: quantification of 14.4 kb (GCR) band, or all high molecular weight species. Error bars; s.d. n=3 (f) Southern blot of normal (9.9 kb) and dicentric (14.4 kb) chromosomes at T= 0, 3.5 and 7 hours after induction of rtf1+. Right: quantification of three experiments. Error bars: s.d. The increased GCRs seen in panel f at T=0 for cdc20-ΔN likely represents increased leakiness of the Purg1 promoter used.
Figure 5
Figure 5. Polδ usage correlates with fragile sites
(a) The positions at which a the small inverted repeat (top) was integrated. Theses correspond to sites around four euchromatic regions that are not associated with a replication origin (open circles), but which showed an intrinsic relative increase in Polδ usage (Polδ:Polε ratio) for the duplex DNA. Blue: relatively increased Polδ, red: relatively increased Polε. Numbers: kb on indicated chromosome (b). Gross Chromosomal Rearrangements (GCRs) resulting from a fork U-turn at the center of the inverted repeat were assayed. Top: a representative Southern blot. Bottom: the % of GCRs (dicentric + acentric as a proportion of total signal) in the population quantified in 3 independent experiments. * = significant p-values (two-tailed Student’s t test): region 1, I-3870/I-3920, 0.037 and I-3890/I-3920, 0.018. Region 3, II-1165/II-1190, 0.010.
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