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Direct repeat recombination downstream of RTS1. (A) Schematic showing the two positions on chromosome 3 where the ade6 − direct repeat recombination reporter (shown in the bottom panel) is inserted downstream of RTS1. (B) Ade + recombinant frequencies for strains MCW7131, MCW7133, MCW7257, MCW7259, MCW7293, and MCW7295. Data are represented as mean ± SD. DOI: 10.7554/eLife.04539.018
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The completion of genome duplication during the cell cycle is threatened by the presence of replication fork barriers (RFBs). Following collision with a RFB, replication proteins can dissociate from the stalled fork (fork collapse) rendering it incapable of further DNA synthesis unless recombination intervenes to restart replication. We use time-la...
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... forks restarted following blockage at RTS1 are prone to perform a U-turn at small inverted repeats positioned within 2.4 kb downstream of the barrier ( Mizuno et al., 2013). To determine whether this property of the restarted fork is due to a tendency for it to collapse and undergo further rounds of recombination, we positioned our ade6 − direct repeat recombination reporter 0.2 kb downstream of RTS1 ( Figure 7A; site A) and measured the frequency of ade + recombinants ( Figure 7B). With RTS1-IO the recombinant frequency was similar to the background level of spontaneous recombination, whereas with RTS1-AO, it increased by ∼134-fold and ∼3.3-fold more than when the barrier is positioned between the ade6 − repeats ( Figure 7B, Table 1). ...
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... forks restarted following blockage at RTS1 are prone to perform a U-turn at small inverted repeats positioned within 2.4 kb downstream of the barrier ( Mizuno et al., 2013). To determine whether this property of the restarted fork is due to a tendency for it to collapse and undergo further rounds of recombination, we positioned our ade6 − direct repeat recombination reporter 0.2 kb downstream of RTS1 ( Figure 7A; site A) and measured the frequency of ade + recombinants ( Figure 7B). With RTS1-IO the recombinant frequency was similar to the background level of spontaneous recombination, whereas with RTS1-AO, it increased by ∼134-fold and ∼3.3-fold more than when the barrier is positioned between the ade6 − repeats ( Figure 7B, Table 1). ...
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... determine whether this property of the restarted fork is due to a tendency for it to collapse and undergo further rounds of recombination, we positioned our ade6 − direct repeat recombination reporter 0.2 kb downstream of RTS1 ( Figure 7A; site A) and measured the frequency of ade + recombinants ( Figure 7B). With RTS1-IO the recombinant frequency was similar to the background level of spontaneous recombination, whereas with RTS1-AO, it increased by ∼134-fold and ∼3.3-fold more than when the barrier is positioned between the ade6 − repeats ( Figure 7B, Table 1). More than 80% of these recombinants are deletions; however, gene conversions also increase substantially by ∼89-fold over spontaneous levels. ...
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... are calculated from the distance between the midpoint of each origin to RTS1 (Siow et al., 2012) and a fork velocity of 3 kb/min, and it is assumed that each origin fires at the same time Figure 6. continued on next page ( Figure 7A; site B) and measured the frequency of ade + recombinants ( Figure 7B and Table 1). ...
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... are calculated from the distance between the midpoint of each origin to RTS1 (Siow et al., 2012) and a fork velocity of 3 kb/min, and it is assumed that each origin fires at the same time Figure 6. continued on next page ( Figure 7A; site B) and measured the frequency of ade + recombinants ( Figure 7B and Table 1). ...
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... switching associated with restarted replication has been shown to decrease with distance from the point of initiation ( Smith et al., 2007;Mizuno et al., 2013), and therefore, it seemed likely that the decline in recombination frequency from site A to site B was a consequence of the maturation of the restarted fork as it progressed from the RTS1 barrier site. However, it was also possible that replication fork convergence was preventing some restarted forks from progressing as far as site B. To investigate this, we measured the recombinant frequency at site B in strains in which ori- 1253 was deleted ( Figure 7B, Table 1). Surprisingly, with RTS1-AO, the frequency of gene conversions increased by 54-fold and deletions by 225-fold compared to RTS1-IO levels, which also represents an overall ∼1.5-fold increase in recombinants compared to site A. These data indicate that restarted forks remain liable to HR over a distance of at least 12.4 kb from their point of initiation with relatively little or no reduction in template switching. ...
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... we have not directly measured the timings of the next phases of RDR (i.e., strand invasion and DNA synthesis), we can estimate an upper limit for the total time that these would take based on the heightened direct repeat recombination at sites A and B downstream of RTS1-AO being an indicator of restarted fork progression (Figure 7). Our observation that deleting ori-1253 dramatically increases recombinant frequency at site B indicates that progression of the restarted fork is constrained by fork convergence. ...
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... observation that deleting ori-1253 dramatically increases recombinant frequency at site B indicates that progression of the restarted fork is constrained by fork convergence. Therefore, the fact that we detect recombination at sites A and B means that some restarted forks reach these sites before the opposing fork emanating from one of the four centromere proximal origins (assuming that at least one of these origins fires in every cell cycle) ( Figure 7A). The most distant of these origins (ori-1141) lies ∼186 kb away from site A and ∼174 kb away from site B, which means that RDR would have up to ∼60 min from the point of fork blockage at RTS1 to reach these sites, assuming a normal replication fork velocity of 3 kb/min and similar firing times for the origins that flank the RFB. ...
Citations
... When a replication fork encounters RTS1 in its blocking orientation, it stalls and reverses but remains unbroken [14][15][16] . The reversed fork is then resolved either by a converging fork or by HR proteins driving its restart through a process termed recombination-dependent replication (RDR) [17][18][19] . RDR commences within~18 min of the fork encountering the barrier and, similar to BIR, exhibits features that distinguish it from canonical DNA replication including a propensity to undergo template switching 17,20-22 . ...
... Neither RDR-associated template switching nor Rad51-mediated multi-invasion are required for Dup-Del formation There are two alternative models to DRFT that could account for Dup-Del formation: 1) RDR-associated template switching ( Supplementary Fig. 1b) 17,19,22 ; and 2) multi-invasion from a reversed replication fork ( Supplementary Fig. 1c) 24 . In the first model, Dup-Del formation could occur if RDR progresses to the H3a sequence adjacent to ura4 and then undergoes a template switch event that relocates the elongating DNA strand to the H3b sequence next to hyg R . ...
... In previous work, we showed that delaying fork convergence by deleting the strong centromere-proximal replication origin, ori-1253, provides more time for the collapsed fork at RTS1 to recruit recombination proteins, be restarted, and progress via RDR towards a downstream ade6direct repeat reporter 17,19,22 . Consequently, this delay results in an increase in Ade+ recombinants due to template switching occurring between the two ade6heteroalleles 17,19,22 . ...
Replication fork stalling can provoke fork reversal to form a four-way DNA junction. This remodelling of the replication fork can facilitate repair, aid bypass of DNA lesions, and enable replication restart, but may also pose a risk of over-replication during fork convergence. We show that replication fork stalling at a site-specific barrier in fission yeast can induce gene duplication-deletion rearrangements that are independent of replication restart-associated template switching and Rad51-dependent multi-invasion. Instead, they resemble targeted gene replacements (TGRs), requiring the DNA annealing activity of Rad52, the 3’-flap nuclease Rad16-Swi10, and mismatch repair protein Msh2. We propose that excess DNA, generated during the merging of a canonical fork with a reversed fork, can be liberated by a nuclease and integrated at an ectopic site via a TGR-like mechanism. This highlights how over-replication at replication termination sites can threaten genome stability in eukaryotes.
... RPA, Rad52 and Rad51 are loaded onto these ssDNA gaps, ensuring fork protection until the arrested fork is either fused with a converging fork or actively restarted by RDR, which occurs approximately 20 minutes after the arrest 6,26-28 . The restarted fork is associated with a non-canonical, mutagenic DNA synthesis in which both strands are synthesized by Pol , making it insensitive to the RFB 26,27,29,30 . ...
Nuclear pores complexes (NPCs) are genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and acting as docking sites for DNA repair. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function occurs independently of SUMO chains formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect RDR dynamics in distinct ways. Our work probes the mechanisms by which the NPC environment ensures optimal RDR.
Highlights
● Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, in a SUMO chain-dependent manner
● Proteasome-associated NPCs foster the progression of restarted DNA synthesis, in a SUMO chain-independent manner
● The nucleoporin Nup60 promotes the spatial sequestration of Ulp1 at the nuclear periphery
● Ulp1 and proteasome activities are differently required for optimal recombination-mediated fork restart.
... However, the expression of hsRAD51-II3A also resulted in the accumulation of collapsed forks, in a more severe way than in the RAD51 depletion condition, suggesting that a stable but unproductive RAD51 filament inhibits alternative fork restart/repair pathways or leads to enzymatic cleavage of stalled forks [36]. Extensively studied in the fission yeast S. pombe, collapsed forks can be restarted by RDR in 15-20 min and can travel over long distances, up to 20 Kb, before fusing with a canonical fork [38][39][40]. During RDR, the DNA synthesis remains semi-conservative but both strands are synthesized by the DNA polymerase delta, likely in an uncoupled manner [39,40]. ...
The perturbation of DNA replication, a phenomena termed "replication stress", is a driving force of genome instability and a hallmark of cancer cells. Among the DNA repair mechanisms that contribute to tolerating replication stress, the homologous recombination pathway is central to the alteration of replication fork progression. In many organisms, defects in the homologous recombination machinery result in increased cell sensitivity to replication-blocking agents and a higher risk of cancer in humans. Moreover, the status of homologous recombination in cancer cells often correlates with the efficacy of anti-cancer treatment. In this review, we discuss our current understanding of the different functions of homologous recombination in fixing replication-associated DNA damage and contributing to complete genome duplication. We also examine which functions are pivotal in preventing cancer and genome instability.
... We have previously developed assays in the fission yeast Schizosaccharomyces pombe for measuring ectopic recombination linked to the initiation of RDR (Fig. 1a) and template switching associated with its elongation phase (Fig. 1b) 30,31 . These assays utilise the polar replication fork barrier (RFB) RTS1, which strongly blocks replication forks and triggers their collapse without inducing a DSB 31,32 . ...
... We have previously developed assays in the fission yeast Schizosaccharomyces pombe for measuring ectopic recombination linked to the initiation of RDR (Fig. 1a) and template switching associated with its elongation phase (Fig. 1b) 30,31 . These assays utilise the polar replication fork barrier (RFB) RTS1, which strongly blocks replication forks and triggers their collapse without inducing a DSB 31,32 . It is thought that the collapsed fork reverses creating a free duplex DNA end, which is processed into a 3′-OH ended ssDNA tail that is ultimately bound by Rad51 31,33,34 . ...
... These assays utilise the polar replication fork barrier (RFB) RTS1, which strongly blocks replication forks and triggers their collapse without inducing a DSB 31,32 . It is thought that the collapsed fork reverses creating a free duplex DNA end, which is processed into a 3′-OH ended ssDNA tail that is ultimately bound by Rad51 31,33,34 . Like in BIR, Rad51 then catalyses the invasion of a homologous duplex DNA, by the ssDNA that it is bound to, generating a D-loop at which new DNA synthesis is primed (Fig. 1a, b). ...
It is thought that many of the simple and complex genomic rearrangements associated with congenital diseases and cancers stem from mistakes made during the restart of collapsed replication forks by recombination enzymes. It is hypothesised that this recombination-mediated restart process transitions from a relatively accurate initiation phase to a less accurate elongation phase characterised by extensive template switching between homologous, homeologous and microhomologous DNA sequences. Using an experimental system in fission yeast, where fork collapse is triggered by a site-specific replication barrier, we show that ectopic recombination, associated with the initiation of recombination-dependent replication (RDR), is driven mainly by the Rad51 recombinase, whereas template switching, during the elongation phase of RDR, relies more on DNA annealing by Rad52. This finding provides both evidence and a mechanistic basis for the transition hypothesis.
... When a replication fork collapses and is unable to resume canonical replication, it can be rescued by a converging fork. However, previous work has shown that a single RTS1 barrier frequently results in HR-dependent replication fork restart even though, in the vast majority of cells, a converging fork would be expected to arrive 27 . This implies that HR-dependent restart occurs irrespective of the status of the locus, but is instead governed by the time it takes to assemble the appropriate machinery. ...
... Using qPCR upstream and downstream of RTS1 we have previously estimated that restart occurs after a delay of~18 min in the vast majority of cells 10 . A separate study demonstrated that HR protein foci begin to appear 10 min after the start of S phase, peak in numbers some 30 min later and that individual foci can remain present for >30 min 27 . To reconcile these two data sets, we have speculated that HR-mediated restart occurs in most cells after a delay of~18 min and that HR proteins remain associated with the locus during HR-restarted replication progress (and possibly during fork convergence and termination with the incoming canonical fork). ...
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.
... Forks arrested at the RFB become dysfunctional and are rescued by opposite forks or, if not available in a timely manner, restarted; both pathways require the binding of Rad51 to the active RFB 6 . Replication fork restart occurs by RDR within ∼20 min and is initiated by an end-resection machinery to generate ssDNA gaps onto which RPA, Rad52, and Rad51 are loaded 4,5,48,49 . RDR is associated with a non-processive DNA synthesis liable to replication slippage and GCRs, during which both strands are synthetized by Polymerase delta, making the progression of restarted forks likely insensitive to the RFB 7,49 . ...
... Consistent with an anchorage to NPCs, the active RFB colocalized with the NP in a less sporadic manner, with interactions lasting for most of the acquisition time in the majority of S-phase cells analyzed. The average time of co-localization was ∼20 min (Fig. 1h), and correlated with the time needed to restart replication forks 48,49 . We conclude that dysfunctional forks transiently anchor to NPCs in S-phase, for a time that coincides with the time needed to complete RDR. ...
Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences.
... When fork progression is blocked at a replication fork barrier (RFB), such as damaged DNA or a protein roadblock, and it cannot be rescued by the convergent fork, for example in regions of low origin density or proximal to telomeres, HR can be used to actively restart and complete replication (Lambert et al. 2005). In S. pombe the replication terminator sequence 1 (RTS1) RFB has been used to study site-specific homologous recombination-restarted replication (HoRReR) (Lambert et al. 2010;Mohebi et al. 2015;Nguyen et al. 2015). Replication termination factor 1 (Rtf1) binds to RTS1 to form a unidirectional RFB that normally functions to control the direction of replication at the mat1 locus to regulate mating-type switching (Dalgaard and Klar 2001). ...
... HoRReR occurs in Sphase and is semi-conservative but utilizes Pol d to synthesize both the leading and lagging strands . This mode of replication is error-prone due to template slippage/switching (Iraqui et al. 2012;Mizuno et al. 2013) that has been suggested to occur due to the frequent engagement of HR proteins catalyzing ectopic recombination (Nguyen et al. 2015). This suggests DNA synthesis during HoRReR is distinct from canonical replication, likely due to an inability to reload CMG following restart. ...
In eukaryotes three DNA polymerases (Pols), α, δ, and ε, are tasked with bulk DNA synthesis of nascent strands during genome duplication. Most evidence supports a model where Pol α initiates DNA synthesis before Pol ε and Pol δ replicate the leading and lagging strands, respectively. However, a number of recent reports, enabled by advances in biochemical and genetic techniques, have highlighted emerging roles for Pol δ in all stages of leading-strand synthesis; initiation, elongation, and termination, as well as fork restart. By focusing on these studies, this review provides an updated perspective on the division of labor between the replicative polymerases during DNA replication.
... Site-specific replication fork barriers, such as the Replication Termination Sequence 1 (RTS1) site in Schizosaccharomyces pombe, have been useful to determine the molecular mechanisms for direct replication fork restart via TS, also referred to as replication-dependent recombination (RDR) or homologous recombination-restarted replication (HoRReR) [102][103][104][105][106][107]. Replication restart at the RTS1 occurs within S phase without the generation of a double-strand break (DSB) [103,107,108], but this type of restart can still be mutagenic [103,109]. ...
... BIR then proceeds by bubble-migration with uncoupled leading and lagging-strand DNA synthesis. A similar process, RDR, occurs at arrested but unbroken RFs (62)(63)(64). In this case, it is likely that the regressed arm of reversed RFs provides the recombination substrate, which is used for in- vasion of the parental duplex ahead of the site of fork reversal with subsequent D-loop DNA synthesis. ...
DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5'-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5'-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2's role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1's ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2's role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.
... When fork progression is blocked at a replication fork barrier (RFB), such as damaged DNA or a protein roadblock, and it cannot be rescued by the convergent fork, for example in regions of low origin density or proximal to telomeres, HR can be used to actively restart and complete replication (Lambert et al. 2005). In S. pombe the replication terminator sequence 1 (RTS1) RFB has been used to study site-specific homologous recombination-restarted replication (HoRReR) (Lambert et al. 2010;Mohebi et al. 2015;Nguyen et al. 2015). Replication termination factor 1 (Rtf1) binds to RTS1 to form a unidirectional RFB that normally functions to control the direction of replication at the mat1 locus to regulate mating-type switching (Dalgaard and Klar 2001). ...
... HoRReR occurs in Sphase and is semi-conservative but utilizes Pol d to synthesize both the leading and lagging strands . This mode of replication is error-prone due to template slippage/switching (Iraqui et al. 2012;Mizuno et al. 2013) that has been suggested to occur due to the frequent engagement of HR proteins catalyzing ectopic recombination (Nguyen et al. 2015). This suggests DNA synthesis during HoRReR is distinct from canonical replication, likely due to an inability to reload CMG following restart. ...
Leading-strand template aberrations cause helicase–polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions ‘on the fly’ to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally. In vitro reconstitution of translesion synthesis–mediated replication fork restart shows how DNA Pol η is recruited to bypass a CPD lesion on the leading strand in the context of the yeast replisome.
