Problems that arise during DNA replication are thought to be the root cause of much of the genome instability, including duplications, deletions and translocations of chromosomal segments, that is associated with cancer and other disease states (Aguilera and García-Muse, 2013; Arlt et al., 2012; Macheret and Halazonetis, 2015). One common problem that arises is accidental collision of the replication fork with proteins that are tightly bound to the DNA (Lambert and Carr, 2013). Such collisions can precipitate a process known as replication fork collapse in which the protein components of the fork (collectively known as the replisome) are thought to partially or completely dissociate from the DNA (Cortez, 2015). In some cases this process may also be associated with DNA breakage, detaching one arm of the replication fork and exposing a single duplex DNA end. Replication fork collapse, either with or without DNA breakage, causes a localized cessation of DNA synthesis that can be remedied through the intervention of homologous recombination (HR) proteins, which restart replication.

In eukaryotes, recombination-dependent replication (RDR) has been studied mostly in the budding yeast Saccharomyces cerevisiae, using experimental systems in which RDR is initiated from a site-specific DNA double-strand break (DSB) (Anand et al., 2013; Kramara et al., 2018; Sakofsky and Malkova, 2017). HR uses intact homologous DNA sequences as templates to repair DSBs and, if such sequences are available for only one side of the break, repair occurs via a RDR process termed break induced replication (BIR). In most cases, BIR has been studied outside of the context of S-phase, with a DSB, rather than a collapsed or broken replication fork, acting as its inducer. However, studies, using a site-specific single-strand DNA break to induce replication fork breakage, have shown that essentially the same BIR process can repair broken forks during S-phase (Mayle et al., 2015).

Similar to other HR-mediated repair processes, BIR involves the 5’ to 3’ nucleolytic resection of a duplex DNA end to provide a single-stranded DNA (ssDNA) tail for the Rad51 recombination protein to load on to (Anand et al., 2013). In yeast, this loading reaction depends on Rad52, which acts as a mediator between ssDNA coated with the ssDNA-binding protein RPA and Rad51 (Krogh and Symington, 2004). Once loaded, Rad51 performs a DNA homology search, followed by strand invasion of the intact DNA sequence that it finds, forming a displacement (D) loop. The D-loop acts as a platform for replication protein reassembly, which seemingly involves most of the main replisome components (Lydeard et al., 2010). However, BIR exhibits a number of features that distinguishes it from canonical replication initiated from a replication origin, including: a requirement for the non-essential DNA Polymerase ∂ subunit Pol32 (Lydeard et al., 2007); a dependence on the Pif1 DNA helicase to promote DNA synthesis (Saini et al., 2013; Wilson et al., 2013); progression via a migrating D-loop or bubble resulting in conservative rather than semi-conservative replication (Saini et al., 2013); a massively increased rate of mutagenesis (Deem et al., 2011); an association with clustered mutations (Sakofsky et al., 2014); and an increased frequency of template switching (TS) (Smith et al., 2007). Given that a BIR-like process has been documented in human cells (Costantino et al., 2014; Roumelioti et al., 2016), it is highly likely that its error-prone nature and propensity for TS causes many of the mutations and genome rearrangements that are associated with cancer and other diseases. It is therefore imperative that the factors that influence BIR’s tendency to cause genomic change are identified, and their conservation of function in related RDR processes assessed, especially in the context of ongoing DNA replication during S-phase.

We, and others, have documented a related process to BIR in the fission yeast Schizosaccharomyces pombe, which is induced by the RTS1 protein-DNA polar replication fork barrier (RFB), as opposed to a DNA break (Lambert et al., 2010; Miyabe et al., 2015; Mizuno et al., 2013; Nguyen et al., 2015). This form of RDR has been termed homologous recombination-restarted replication (HoRReR) (Miyabe et al., 2015) but, in this paper, we will refer to it simply as RDR. Replication forks collapse, and recruit HR proteins, within approximately 10 min of collision with RTS1 (Mohebi et al., 2015; Nguyen et al., 2015). Similar to BIR, Rad51 and Rad52 promote replication restart at RTS1 and, in doing so, can inadvertently cause genomic rearrangements through the recombination of repetitive DNA elements (Ahn et al., 2005; Lambert et al., 2005; Lambert et al., 2010; Mizuno et al., 2009; Morrow et al., 2017; Nguyen et al., 2015). Also like BIR, RDR induced by RTS1 is highly mutagenic and prone to TS (Iraqui et al., 2012; Mizuno et al., 2013; Nguyen et al., 2015). In this paper, we advance from our earlier study by identifying some of the key factors that influence TS during RDR, including key antirecombinogenic DNA helicases and collisions between restarted replication and tRNA transcription. We also highlight apparent similarities and differences between TS associated with BIR in S. cerevisiae and RTS1-induced RDR in S. pombe.

The factors affecting TS associated with recombination-mediated DNA replication have hitherto been studied mainly in the context of BIR in S. cerevisiae and outside of S-phase. In our study we have measured TS associated with RDR from a collapsed, yet unbroken, replication fork in S. pombe. With this system, we have identified similarities with BIR in S. cerevisiae, as well as some potential differences. To place our findings into a conceptual framework, we will first discuss a hypothetical model for TS, which is elaborated from a previous model (Figure 7) (Nguyen et al., 2015). We envisage that the collapsed replication fork regresses to form a classic ‘chicken foot’ structure with an exposed ssDNA tail formed by the nucleolytic activity of the Mre11-Rad50-Nbs1-Ctp1 complex and Exo1 (Figure 7, step 1) (Teixeira-Silva et al., 2017). Rad51 loads onto the ssDNA tail to form a nucleoprotein filament that first locates a homologous duplex and then invades it to form a D-loop (Figure 7, step 2). Pol∂ extends the 3’ end of the invading strand (Miyabe et al., 2015) and, as it does, the D-loop moves in tandem (Figure 7, step 3) (Saini et al., 2013). Although not shown in Figure 7, lagging strand DNA synthesis may be established on the nascent leading strand giving rise to conservative replication, which could then be directed into semi-conservative replication by branch migration of the regressed replication fork behind the D-loop (Nguyen et al., 2015). Such a model would be compatible with the finding that RDR, triggered by fork collapse at RTS1, is semi-conservative (Miyabe et al., 2015), and contrasts with BIR, which is conservative (Donnianni and Symington, 2013; Saini et al., 2013). As DNA replication progresses, the associated migrating D-loop is likely to be susceptible to dissociation, either through the action of DNA helicases and/or by branch migration of the regressed replication fork ‘catching up’ with it (Nguyen et al., 2015). Dissociation of the D-loop in a region of repetitive DNA would lead to TS if the ejected DNA strand re-invades the ‘wrong’ repeat (Figure 7, steps 4 and 5; note that the DNA repeats, in yellow, are directly orientated as they are in our genetic assay). RDR is terminated upon encounter with the oncoming replication fork (Figure 7, steps 6 and 7) and, during this process, the invading DNA strand may either be ligated to the nascent lagging strand of the oncoming fork (Figure 7, step 7a) or be unwound (Figure 7, step 7b). If unwound, a second TS may be triggered (not shown) or the regressed replication fork may simply re-set by branch migration and merge with the oncoming fork (Figure 7, steps 8b and 9b). Note that these events can give rise to a gene conversion in our genetic assay (Figure 1B). Ligation of the invading DNA strand to the nascent lagging strand of the oncoming fork would prevent further TS and consolidate the formation of a deletion, with branched DNA structures being resolved by nucleases (not shown) (Lorenz et al., 2009) or by the completion of DNA replication (Figure 7, steps 8a and 9a). In the latter case, the region being deleted would form a heterologous loop of ssDNA (Figure 7, step 9a), which could be removed by a large DNA loop repair pathway (Clikeman et al., 2001; Sommer et al., 2008).

Figure 7

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TS associated with BIR in S. cerevisiae is thought to reflect an inherent instability of the migrating D-loop, which has evolved to encourage DSB repair by synthesis-dependent strand annealing (Smith et al., 2007). The observation that TS in S. cerevisae is constrained to the first 10 kb of template DNA that is copied, suggests that the D-loop eventually matures into a more stable structure (Mayle et al., 2015; Smith et al., 2007). This maturation process may involve cleavage of the D-loop by structure-specific nucleases such as Mus81 (Mayle et al., 2015; Osman and Whitby, 2007). Our finding that TS downstream of RTS1-AO is generally biased in favour of deletions, whereas at the RFB gene conversions predominate, suggests that the frequency of double TS events declines soon after replication has restarted. Interestingly, Carr and colleagues reported that the propensity for restarted replication to perform a U-turn at small inverted repeats positioned downstream of RTS1 declined 4-fold when the distance between RFB and repeats was increased from 0 to 2 kb, and then appeared to plateu (Mizuno et al., 2013). These observations suggest that the putative migrating D-loop, which is formed following replication fork collapse at RTS1-AO, undergoes partial stabilization shortly after it is formed. However, unlike with BIR in S. cerevisiae, a degree of D-loop instability must be retained because we can detect TS over distances of at least 75 kb downstream of the site of fork collapse. Our finding, that tRNA^GLU08HO increases TS downstream of RTS1-AO, suggests that head-on collisions with transcription complexes might exacerbate D-loop instability and be a key trigger in promoting TS.

The apparent disparity in D-loop maturation, between BIR in S. cerevisiae and RDR in S. pombe, may reflect differences in D-loop processing. In S. cerevisiae, Mus81 constrains TS during BIR presumably by resolving the D-loop into a more stable fork structure (Mayle et al., 2015). However, in S. pombe, we found no evidence that Mus81 is required to constrain TS during RDR. In fact the only constraint, over the region in which TS can occur in S. pombe, seems to be the point at which the restarted replication converges with the oncoming replication fork - a property which is shared with BIR in S. cerevisiae (Mayle et al., 2015; Nguyen et al., 2015).

TS associated with restarted replication is thought to be responsible for generating many of the genome rearrangements that are associated with cancer and other disease states (Carvalho and Lupski, 2016; Hastings et al., 2009). However, some complex genome rearrangements can span distances of hundreds of kilobases and, therefore, could not be formed from a single restarted replication event if TS was limited to a 10 kb region downstream of the site of fork collapse (Hastings et al., 2009). Our finding, that TS events occur over a much greater distance in S. pombe than they do for BIR in S. cerevisiae, shows that TS, associated with the repair of a single collapsed replication fork, may be capable of generating some of the long-range and complex genome rearrangements seen in humans.

Similar to BIR in S. cerevisiae, the Pif1 family DNA helicase Pfh1 promotes RDR, which explains why there is a general reduction in the levels of TS downstream of RTS1-AO in pfh1-m21 and -mt* mutants. In S. cerevisiae, Pif1 aids DNA synthesis during BIR by driving D-loop migration (Wilson et al., 2013), and Pfh1 might do likewise during RDR in S. pombe. Moreover, our observation, that resected regressed replication forks accumulate at RTS1-AO in a pfh1-mt* mutant, suggests that Pfh1 is a candidate for promoting the putative branch migration and/or re-setting of the regressed replication fork behind the migrating D-loop (see Discussion above). Pfh1 also has the ability to drive DNA replication by dislodging protein-DNA barriers via its so-called sweepase activity (Sabouri et al., 2012; Steinacher et al., 2012). Efficient RDR past RTS1 presumably requires removal of the barrier protein Rtf1, which may depend on Pfh1’s sweepase activity.

Despite Pfh1 being needed for efficient RDR, we were able to detect an increase in TS in a pfh1-m21 mutant if we provided more time for replication restart to occur, which shows that Pfh1, in addition to promoting RDR, also plays a role in suppressing TS. This finding contrasts with BIR in S. cerevisisae where Pif1 has no affect on TS (Stafa et al., 2014). Unlike S. pombe and humans, which have only one Pif1 family helicase, S. cerevisiae has a second Pif1 helicase called Rrm3, which, whilst seemingly not required for BIR, is important for the repair of replication-associated DSBs as well as replication past stable protein-DNA complexes (Ivessa et al., 2003; Muñoz-Galván et al., 2017; Wilson et al., 2013). It is possible that Pif1 and Rrm3 act redundantly in the suppression of TS in S. cerevisiae. Alternatively, the role of Pif1 helicases in suppressing TS may only be evident when RDR acts during S-phase alongside ongoing canonical DNA replication. Specifically, they may play an important role in directing the termination of DNA replication where a canonical fork encounters a D-loop. We speculate that Pfh1 drives D-loop dissociation during termination through its action as an accessory helicase at the oncoming DNA replication fork, where it likely translocates on the lagging template strand in a 5’ – 3’ direction (i.e. towards the D-loop and on the same DNA strand that the invading strand is annealed to) (Sabouri, 2017) (Figure 7, step 7b). Without this activity there would be a greater tendancy for the 3’ end of the invading strand at the D-loop to ligate to the lagging strand of the oncoming fork, which would specifically favour deletion formation in our genetic assay (Figure 7, steps 7a – 9a). Indeed, in the instances where we can detect an increase in Ade⁺ recombinants when pfh1 is mutated, it is specifically deletions that increase, with the frequency of gene conversions either decreasing or remaining unchanged (Figure 2, Figure 2—figure supplement 1, Figure 4, Figure 4—figure supplement 1).

In addition to Pfh1, we have identified a further three conserved DNA helicases that suppress TS downstream of RTS1-AO, namely Rqh1, Srs2 and Fbh1. These helicases, and their homologues in other organisms, are well known as major players in directing the outcome of homologous recombination reactions (Doe and Whitby, 2004; Fugger et al., 2009; Kohzaki et al., 2007; Larsen and Hickson, 2013; Lorenz et al., 2009; Morishita et al., 2005; Niu and Klein, 2017; Osman et al., 2005; Simandlova et al., 2013; Tsutsui et al., 2014). However, their role in suppressing TS downstream of a collapsed replication fork has not previously been demonstrated, though it has been suggested that the homologue of Rqh1 in S. cerevisiae (Sgs1) might limit translocations between highly diverged genes by suppressing TS during BIR (Schmidt et al., 2006). Based on the known activities of their homologues, Rqh1, Srs2 and Fbh1 have the potential to influence TS in various ways. For example, Sgs1 and its human homologue BLM, as well as S. cerevisiae Srs2, are capable of dissociating D-loops in vitro (Bachrati et al., 2006; Bugreev et al., 2007; Dupaigne et al., 2008; Fasching et al., 2015; Liu et al., 2017; van Brabant et al., 2000). The dissociation of a nascent D-loop can abort a TS event before a genetic change is made (e.g. a deletion or gene conversion in our genetic assay) and, therefore, the loss of this activity might account for the increase in TS we observe in both rqh1∆ and srs2∆ mutants (Figure 5). Indeed, Lambert and colleagues showed that Rqh1 was responsible for the disappearance of recombination intermediates at RTS1 in cells lacking CAF1, and proposed that this was due to its ability to drive D-loop dissociation (Pietrobon et al., 2014).

Another way in which Rqh1 may affect TS is by catalyzing the branch migration of the regressed replication fork behind the migrating D-loop. The regressed fork forms a four-way/Holliday junction, which is a structure that several RecQ–type helicases have been shown to unwind/branch migrate in vitro (Cejka and Kowalczykowski, 2010; Karow et al., 2000; LeRoy et al., 2005; Machwe et al., 2011). As discussed above, branch migration of the four-way junction into the ‘back’ of the D-loop would cause the D-loop to unwind. This might happen more frequently during an attempted TS event if the four-way junction continues to migrate whilst the D-loop is being re-established.

In S. cerevisiae, Rad51 promotes strand re-invasion, leading to a TS event during BIR (Anand et al., 2014). Presumably, Rad51 loads onto the ejected DNA strand following dissociation of the migrating D-loop to form a nucleoprotein filament. Both Srs2 and Fbh1 regulate nucleoprotein filament formation by dissociating Rad51 from DNA (Krejci et al., 2003; Simandlova et al., 2013; Tsutsui et al., 2014; Veaute et al., 2003). The loss of this anti-recombinogenic activity might account for the increase in TS observed in srs2∆ and fbh1∆ mutants.

Whilst dissociation of a nascent D-loop might prevent TS recombination, the unwinding of a fully established migrating D-loop would promote it. Indeed, this is exactly the activity that Mph1 is thought to catalyze during BIR in S. cerevisiae (Stafa et al., 2014). Surprisingly, we found that Fml1, despite promoting ectopic recombination during the restart of replication, appeared to have no effect on TS once replication had restarted. It is possible that a reduction in TS caused by lower levels of migrating D-loop dissociation is offset by an increased number of D-loops reaching our 12.4 kb genetic reporter. We are currently developing methods to measure restarted replication tract lengths, which will allow us to better calibrate our TS data.

In conclusion, we have identified a number of factors that influence TS associated with replication restart from a collapsed replication fork, revealing both similarities and potential differences with TS during BIR in S. cerevisiae. The fact that S. pombe and S. cerevisiae are only distant relatives, being about as divergent from each other as each is to animals (Heckman et al., 2001; Sipiczki, 2000), means that the similarities we have reported (e.g. Pif1 helicases promoting replication restart) are likely to be conserved in humans. Even the apparent disparities may simply reflect differences in the recombination assays used rather than fundamental mechanistic differences between RDR in S. pombe and S. cerevisiae. This is exemplified in S. cerevisiae, where the importance of Mus81 in limiting TS during BIR appears to depend on the recombination assay (Mayle et al., 2015; Stafa et al., 2014). In future studies, it will be important to establish whether the apparent disparities between RDR in S. pombe and BIR in S. cerevisiae reflect real species-specific differences, fundamental differences in the restart of replication from a DSB/broken replication fork versus a collapsed, yet unbroken, fork or simply differences in the recombination assays that have been used.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Manisha Jalan

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Present address

   Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4467-4934

2. Judith Oehler

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8397-6492

3. Carl A Morrow

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Present address

   Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

4. Fekret Osman

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   Formal analysis, Supervision, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

5. Matthew C Whitby

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   matthew.whitby@bioch.ox.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0951-3374

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