Homologous recombination and Mus81 promote replication completion in response to replication fork blockage

Article Homologous recombination and Mus81 promote replication completion in response to replication fork blockage Benjamin Pardo 1,2,* , María Moriel-Carretero 1,2,† , Thibaud Vicat 1 , Andrés Aguilera 2,** & Philippe Pasero 1 Abstract Impediments to DNA replication threaten genome stability. The homologous recombination (HR) pathway has been involved in the restart of blocked replication forks. Here, we used a method to increase yeast cell permeability in order to study at the molecular level the fate of replication forks blocked by DNA topoisomerase I poisoning by camptothecin (CPT). Our results indicate that Rad52 and Rad51 HR factors are required to complete DNA replication in response to CPT. Recombination events occurring during S phase do not generally lead to the restart of DNA synthesis but rather protect blocked forks until they merge with convergent forks. This fusion generates structures requiring their resolution by the Mus81 endonuclease in G 2 /M. At the global genome level, the multiplicity of replication origins in eukaryotic genomes and the fork protection mechanism provided by HR appear therefore to be essential to complete DNA replication in response to fork blockage. Keywords BIR; fork restart; Mus81; recombination; replication Subject Categories DNA Replication, Recombination & Repair DOI 10.15252/embr.201949367 | Received 26 September 2019 | Revised 16 April 2020 | Accepted 20 April 2020 | Published online 17 May 2020 EMBO Reports (2020)21:e49367 Introduction Chromosome duplication during the S phase is a crucial step of cell division. DNA replication in eukaryotes is initiated from multiple origins distributed on all chromosomes. Replication forks progress along chromosomes arms until merging with another incoming fork at termination regions. The progression of replication forks can be hindered by obstacles in the DNA template such as DNA lesions. Genome stability is particularly at risk when damaged DNA molecules are replicated. Failure in DNA damage repair can lead to the terminal arrest or breakage of replication forks and ultimately to the distribution of under-replicated and/or broken chromosomes to the daughter cells after mitotic division. When a fork becomes dysfunctional, the completion of replication could be ensured by a converging functional fork, a process that can be favored by the firing of nearby dormant origins [1,2]. Alternatively, dysfunctional replication forks could also be restarted, a mechanism that requires the homologous recombination (HR) pathway [3–5]. During HR, Rad52 and Rad51 are fundamental to coat the ssDNA generated by resection and carry out the strand invasion and exchange reactions [6]. At dysfunctional forks, Rad51 loading by Rad52 also regulates nascent DNA strands degradation by exonucleases [7–9]. Whether this protection mechanism is different from the restart mechanism has not been clearly determined [7,10–12]. Recombination-mediated replication restart has been mainly studied in the yeast cellular model using site-specific tools to break or block replication forks at a unique location [3,4,13–17]. Replication restart in Saccharomyces cerevisiae has been recently investigated using a Flp-nick system, which produces a site-specific DNA nick that is converted into a one-ended DSB upon the passage of a replication fork [4,14,18]. Recombination-mediated restart seems to occur by break-induced replication (BIR), an HR pathway mainly characterized outside of S phase [19,20]. BIR is favored when only one DSB end is able to invade a homologous template. In that case, DNA synthesis primed within the displacement loop (D-loop) can proceed to the chromosome end by a conservative mechanism [21– 24]. In particular, the non-essential DNA polymerase dsubunit Pol32 is required for priming DNA synthesis inside the D-loop and the Pif1 DNA helicase is required to promote its migration [24–26]. Lastly, BIR synthesis is highly mutagenic [4,27] and subjected to template switching, leading to complex chromosomal rearrangements [28–31]. Terminally blocked replication forks at the RTS1 barrier in Schizosaccharomyces pombe can be restarted by HR without DSB formation [3,7,32–35]. Similar to BIR in S. cerevisiae, replication restart at RTS1 requires the DNA polymerase dand the Pfh1 DNA helicase, is highly mutagenic and prone to template switching [35–38]. In that context, HR is not thought to be primed from a DSB 1Institut de Génétique Humaine, Université de Montpellier-CNRS, Montpellier, France 2Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville, Spain *Corresponding author. Tel: +33 434359943; E-mail: [email protected]nrs.fr **Corresponding author. Tel: +34 954468372; E-mail: [email protected] † Present address: Centre de Recherche en Biologie cellulaire de Montpellier, Université de Montpellier-CNRS, Montpellier, France ª2020 The Authors EMBO reports 21:e49367 |2020 1of 19 but from the tip of a reversed fork, which results from the displacement and reannealing of the nascent strands together [32,39,40]. It remains to be determined whether replication restart studied at locus-specific barriers occurs in the same way at the genome-wide level in response to natural replication impediments. For example, the DNA topoisomerase 1 (Top1) normally introduces a transient nick to relax supercoiled DNA during transcription and replication [41]. During this reaction, Top1 remains covalently attached to the 30end of the break, forming a “cleavage complex” (Top1cc), before the relaxation of DNA and religation of the break. Cells are constantly challenged with blocked Top1ccs, which is a major driver of mutagenesis in highly transcribed genes [42,43]. Furthermore, cells devoid of both Tdp1 and Wss1, two factors involved in the removal of Top1ccs, have a severe Top1-dependent growth defect [44,45], which indicates that Top1ccs are natural threats to cell survival. Top1ccs can also be stabilized by the drug camptothecin (CPT) [46]. CPT is thought to cause the formation of a one-ended DSB, the typical substrate for BIR, upon the collision of the replication fork with the nick in the Top1cc, thus inducing a replication stress [47–49]. However, CPT-induced DSBs have not been observed in yeast cells [50,51]. More recently, it has been shown that CPT treatment of yeast cells induces fork reversal [50,52], as a possible consequence of accumulation of positive supercoils ahead of replication forks due to Top1 inhibition [53]. Hence, replication fork blockage by CPT-stabilized Top1ccs appears as a relevant model to address whether replication restart occurs at the global genomic level. Finally, it remains unclear which factors participate in the resolution of the recombination intermediates during HR-mediated restart of DNA replication. The structure-selective endonuclease Mus81 has been proposed to cleave either blocked or reversed forks to promote repair by HR [54–56]. However, Mus81 is not required for replication restart at RTS1 in fission yeast, nor for the repair of a replication-born DSB in budding yeast [3,17]. Mus81 is nevertheless involved in the processing of these recombination events, as they accumulate in its absence [3,17], resulting in a decreased amount of final repair products [17,57]. By using the Flp-nick system, it has been proposed that Mus81 limits replication restart by Pol32-depen- dent BIR in S phase by processing the migrating D-loop [4]. Mus81 catalytic activity is normally very low in S phase and only increases at the G 2 /M transition, when the Mms4 regulating subunit of the complex is hyper-phosphorylated by multiple kinases [58–61]. Hence, the role of Mus81 in replication restart by HR in S phase appears contradictory to the regulation of its activity. We took advantage of our study to clarify the role of Mus81 in recombination-mediated restart of DNA replication. Our data reveal that HR is primarily providing a replication fork protection mechanism rather than promoting the restart of DNA synthesis from blocked forks. Thanks to a method to increase CPT entry into yeast cells, we show that S phase completion in response to Top1 poisoning requires Rad52 and Rad51. We propose that HR promotes the formation of a D-loop structure that protects blocked forks until they merge with converging forks. Completion of replication also requires Mus81 in G 2 /M to promote the termination of DNA replication at protected forks. We confirmed our results obtained in CPT-treated cells by using an independent system in which a specific mutation of the Rad3 DNA helicase generates a similar replication stress. This allowed us to demonstrate that the mechanism we have characterized upon Top1 poisoning is independent of the accumulation of supercoiled DNA and DNA-protein crosslinks. The engagement of HR to protect blocked forks and to ensure the completion of DNA replication appears therefore as a general mechanism to respond to replication stress. Results Top1poisoning by CPT and the rad3-102 allele exert a genetically similar replication stress In this study, we have chosen to study the dynamics of DNA replication challenged by CPT treatment, which exacerbates at the genome-wide level the presence of trapped Top1 on the DNA template. Resistance to CPT-induced DNA damage absolutely requires the homologous recombination (HR) machinery, as null mutations of RAD52, MRE11,RAD50, and XRS2 lead to the highest sensitivities to low doses of CPT compared to wild-type cells (Fig 1A) [62,63]. rad51Δmutants are also highly sensitive to low CPT doses but to a lesser extent than its upstream regulator Rad52 (Fig 1B and C, Appendix Fig S1A) [4]. Interestingly, Rad52 and the members of the MRX complex (Mre11-Rad50-Xrs2), but not Rad51, are essential for the survival of cells bearing the rad3-102 mutation: This mutation impairs the nucleotide excision repair (NER) pathway by increasing the binding of the TFIIH complex to a single-strand DNA gap intermediate, which prevents its subsequent filling [64,65]. Additionally, it has been described that the elevated CPT sensitivity of a nuclease-deficient Mre11 mutant (mre11-3) can be suppressed by the absence of the Ku complex and in an Exo1- dependent manner (Appendix Fig S1B) [66]. This suggests that Mre11 counteracts the action of Ku at CPT-induced DSBs ends, which limits Exo1-dependent DNA resection. Similarly, although the combination of mre11-3 with rad3-102 is not lethal, it sensitizes the cells to UV exposure, a context in which replication stress is enhanced in the rad3-102 background [65] (Fig 1D). Strikingly, the increased UV sensitivity of rad3-102 mre11-3 compared to rad3-102 can be partially suppressed by the absence of Ku in an Exo1-depen- dent manner (Fig 1D and Appendix Fig S1C). These data show that Mre11, Ku, and Exo1 play similar roles in the repair of DNA damage induced by rad3-102 or CPT. Finally, rad3-102 has been found lethal in combination with both rad51Δand pol32Δ, leading to the proposal that DNA repair in the absence of Rad51 in these cells is backed up by the presence of the Pol32 non-essential subunit of the DNA polymerase @[65,67]. Remarkably, we found that the rad51Δ pol32Δdouble mutant was more sensitive to CPT than the rad51Δ single mutant (Fig 1B and C), suggesting that, as in rad3-102 cells, Pol32 also partially compensates the absence of Rad51 to cope with CPT-induced DNA damage. Thus, rad3-102 cells suffer from a replication stress that mimics the effect of CPT. However, accumulation of topological stress is not expected in rad3-102 cells, since they are not affected in DNA supercoils removal by Top1. Neither Tdp1 (tyrosyl-DNA phosphodiesterase 1) nor the metalloprotease Wss1 are required for survival in rad3-102 cells, consistent with the TFIIH complex not being covalently linked to DNA (Appendix Fig S1D). Overall, in view of the strikingly similar genetic requirements for the cell survival in response to CPT and in the rad3-102 background, we decided to use 2of 19 EMBO reports 21:e49367 |2020 ª2020 The Authors EMBO reports Benjamin Pardo et al A B D EF C Figure 1. ª2020 The Authors EMBO reports 21:e49367 |2020 3of 19 Benjamin Pardo et al EMBO reports these two systems to further investigate the repair mechanisms required to cope with this type of replication stress, independently from the accumulation of DNA supercoiling and DNA-protein crosslinks. Rad51 and Pol32 independently promote DSB repair by break-induced replication First, we asked what could be the specific contribution of Pol32 to DNA repair in the absence of Rad51. We previously proposed that Pol32 may stabilize strand invasion in the absence of Rad51 by promoting the priming of DNA synthesis [67], further supported by the essential role of Pol32 for viability of cells lacking Rad51 that are defective in histone H3K56 deacetylation [68]. Among DSB repair pathways mediated by HR, Pol32 has only an essential role during BIR. In the absence of Pol32, the initiation of DNA synthesis during BIR is compromised [25]. Moreover, the recovery of both Rad51-dependent and Rad51-independent survivors in cells lacking telomerase, thought to occur by BIR, also relies on Pol32 [25]. Finally, the BIR pathway has been proposed to be involved in the repair of broken replication forks [4]. We thus wondered whether Pol32 compensatory function in the absence of Rad51 in cells exposed to CPT or in the rad3-102 background could be related to its function in BIR. To assess the redundant role of Pol32 over Rad51 in BIR, we used a well-described chromosomal system [23] in which a single DSB is induced by the HO endonuclease. In this system, only one of the two ends can undergo homology-dependent strand invasion at an ectopic location. Subsequent priming and elongation of DNA synthesis reaching the chromosome end lead to the production of viable Lys2 + recombinants (Fig 1E). In this system, the absence of Rad52 decreased the BIR frequency by about three orders of magnitude compared to the wild type (Fig 1F). Deletion of RAD51 or POL32 also significantly decreased the BIR frequency compared to the wild type (Fig 1F) [23]. However, only the combined absence of Rad51 and Pol32 did affect the BIR frequency as much as in the absence of Rad52 (Fig 1F). These results show that Pol32 can promote BIR in the absence of Rad51 and indicate that Pol32 functions in BIR to facilitate both strand invasion and priming of extensive DNA synthesis. The absence of Pol32 alone affects cell survival only in response to high CPT concentrations (Fig 1B) [69]. Altogether, these results suggest Pol32-dependent BIR-mediated synthesis is not the main pathway required to cope with CPT-induced DNA damage and that the role of Pol32 in the absence of Rad51 may be ascribed to its function in facilitating strand invasion during HR. Mus81 is involved in HR-mediated repair involving both Rad51 and Pol32 In human cells, the structure-specific endonuclease (SSE) Mus81 has been shown to generate DSBs in response to CPT treatment, leading to the suggestion that Mus81 may cleave stalled or reversed replication forks upon Top1 poisoning to promote fork restart [54]. In budding yeast, null and catalytically dead (mus81-dd) mutations of MUS81 have been shown to sensitize cells to mild CPT doses (Fig 2A) [62,63,70–72]. The absence of Mus81 is backed up by the Yen1 SSE, as shown by the higher CPT sensitivity of the double mus81Δyen1Δmutant compared to mus81Δ(Fig EV1A) [71,72]. Moreover, Mus81 and Yen1 have been proposed to limit BIR synthesis initiated from a replication-born DSB [4]. We asked whether Mus81 could have a similar role in CPT- induced DNA damage repair. First, we observed that the combination of mus81Δwith rad51Δand pol32Δmutations resulted in an increased CPT sensitivity than either single mutants (Fig 2B) [4], suggesting that Mus81 participates in Rad51- and Pol32-dependent repair of DNA damage induced by CPT. As for, rad51Δ,mus81Δ,or mus81-dd are not lethal in combination with rad3-102 but the double mutant cells exhibited a much higher sensitivity to UV than single mutant cells (Fig 2C) [65]. We analyzed the redundancy between Mus81 and Yen1 for survival in the rad3-102 background. rad3-102 yen1Δcells were not more sensitive to UV than rad3-102 cells (Fig EV1B), consistent with yen1Δcells not being sensitive to CPT (Fig EV1A). The rad3-102 mus81Δyen1Δtriple mutants were unviable (Fig EV1C), indicating that Yen1 also backs up Mus81 in rad3-102 cells. Finally, we found that rad3-102 mus81Δpol32Δ mutants were unviable and rad3-102 mus81Δrad51Δcells had a severe growth defect (Fig 2D). These results indicate that Mus81 is required for repair mediated by both Rad51 and the polymerase d subunit Pol32 in rad3-102 cells. Mus81 and Pol32 are not required for replication progression upon Top1poisoning Next, we reasoned that if an increased CPT sensitivity is associated with replication defects, we should observe an impaired progression through S phase. Opposite to other cellular models, it was reported that an acute exposure to CPT in liquid cultures of S. cerevisiae cells did not induce a delay in S phase progression but rather a prolonged arrest in G 2 /M [50,51,73]. CPT is highly insoluble in culture media, and the water-soluble derivatives of CPT (topotecan and irinotecan) affect neither yeast cell growth nor their survival [74], thus not ◀Figure 1. CPT-treated or rad3-102 cells share the same genetic requirements to cope with DNA damage. A, B CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed. C Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values SD are plotted. Four biological replicates have been performed. D UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed. E Schematic representation of the BIR assay. Two fragments of the LYS2gene sharing 2.1kb of homology (lys and ys2) were integrated on chromosomes V and XI, respectively. Induction of HO endonuclease expression under control of the GAL1promoter produces a DSB next to the lys fragment that can be repaired by BIR. BIR events can be scored by selecting survivor colonies, which harbor a functional LYS2gene. F BIR frequencies (Lys + survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean SD from 3to 6biological replicates. ns, non-significant difference, *P=0.0134, Mann–Whitney unpaired t-test. 4of 19 EMBO reports 21:e49367 |2020 ª2020 The Authors EMBO reports Benjamin Pardo et al being exploitable for our study. We suspected a permeability issue for CPT entry into the cells, as described for the proteasome inhibitor MG132 and the DNA polymerase inhibitor aphidicolin [75,76]. We therefore used a modified culture medium described to render cells more permeable to the antifungal agent brefeldin A [77] in order to increase the cell permeability to CPT. We took advantage of the natural blue fluorescence emitted by the CPT compound when excited by 350 nm UV light to quantify the relative amount of CPT inside cells. Incubation of an asynchronous cell culture with CPT for 30 min in MPD +SDS medium (minimal-proline-dextrose +0.003% SDS) [77] allowed the detection of blue fluorescence inside cells, whose quantification was up to 10 times higher than in cells incubated with CPT for 30 min in standard MAD medium (minimalammonium-dextrose) (Fig EV2A and B). We then used these conditions to analyze the progression through a single S phase in the presence of CPT by flow cytometry. Wild-type cells were synchronized in G 1 with a-factor, incubated with CPT for 1 h and then released from G 1 into S phase, still in the presence of CPT. As observed by others, CPT did not alter S phase progression when cells were grown in normal MAD medium (Fig EV2C) [50,51,73], but when incubated with CPT in MPD +SDS medium, they progressed significantly slower through S phase than control wild-type cells incubated with DMSO, thus reaching the G 2 /M phase later (Fig EV2C). Of note, the addition of SDS in the culture medium did not perturb the G 1 -S transition, as previously reported with higher concentrations of SDS [78]. Thus, these experimental conditions allowed us to perform the first characterization of the effect of CPT on DNA replication using S. cerevisiae cells as a model system. Using these conditions, we next asked how the HR factors Rad52 and Rad51, the Mus81 resolvase, and the polymerase dsubunit Pol32 contribute to the progression of replication forks encountering trapped Top1. At the global level, using flow cytometry, we could observe that the S phase delay was strikingly increased in CPT- treated cells lacking one of the two main HR factors, Rad52 or Rad51, compared to wild-type cells (Fig 3A). However, in the absence of Mus81 or Pol32, S phase progression was as affected by CPT as in wild-type cells. We confirmed these results using the rad3-102 mutation. We synchronized cells in G 1 phase with a-factor and irradiated them with UV-C. Cells were kept for 2 h in G 1 before release into S phase, in order to let compromised NER leave DNA gaps or nicks bound by the TFIIH complex in the rad3-102 mutant, as described previously [65]. As expected, UV irradiation only affected S phase progression in rad3-102 cells and to a greater extent in rad3-102 cells lacking the HR factor Rad51 (Fig 3B). However, rad3-102 cells lacking Mus81 or Mus81 nuclease activity did not show an increased S phase delay compared with rad3-102 single mutants (Fig 3C). All these results indicate that HR factors Rad52 and Rad51 are required to promote S phase progression when replication stress is induced by CPT. However, neither Mus81 nor Pol32 is required for bulk DNA synthesis in these conditions. To confirm these data at the molecular level, we analyzed nascent DNA synthesis by DNA combing from asynchronous cell cultures treated with CPT. DNA combing allows monitoring replication fork progression genome-wide though at the level of individual DNA molecules [79,80]. After incubation with CPT for 2 h, cells were pulse-labeled with the thymidine analog EdU for 20 min before combing analysis (Fig 4A). We first compared the length of the EdU tracks in all strains under unchallenged conditions (+DMSO). Only mus81Δcells showed a significant decrease of the EdU track length compared to wild-type cells (Fig 4A; P=0.0015). This result agrees with a recent report that proposed a role for human Mus81 in DNA replication in the absence of exogenous damage [81]. Then, we focused our attention onto the effect of CPT on fork progression in each strain. As expected from cell cycle analyses (Fig 3A), CPT treatment significantly affected fork progression in all strains compared to the DMSO control (Fig 4A). The EdU track length of wild-type cells was reduced by 47% and that of mutant cells was further reduced to 55% in rad51Δcells and 75% in rad52Δcells (Fig 4A). However, the reduction in fork progression caused by CPT in mus81Δ(45%) and in pol32Δ(37%) cells was more similar to that of wild-type cells (Fig 4A). To accurately evaluate the effect of CPT in mutants compared to the wild type, we calculated a conversion factor to equal the median of each DMSO-treated samples from mutants to that of the wild type. We then used this normalization factor for each mutant to convert all track length values of CPT- treated samples. The statistical analysis of the normalized data shows that only rad52Δand rad51Δmutants are more affected by CPT than wild-type cells (Fig EV3A). Our results thus argue against a role of Mus81 in replication restart in CPT-treated cells, as was suggested in human cells [54]. This was confirmed in rad3-102 A B C D Figure 2. Mus81 is required in the restart pathway involving both Rad51 and Pol32. A, B CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed. C UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed. D Synthetic combinations of rad3-102 mus81Δwith rad51Δand pol32Δ. Tetrads dissected on YPAD medium are shown. Triangles indicate either a severe growth defect or lethality. ª2020 The Authors EMBO reports 21:e49367 |2020 5of 19 Benjamin Pardo et al EMBO reports A B C Figure 3. Mus81 is not required for S phase progression in CPT-treated and rad3-102 cells. A Analysis of DNA content by flow cytometry of G 1 phase synchronized wild-type, rad52Δ,rad51Δ,mus81Δ,and pol32Δcells and further released into S phase. Cells were synchronized in G 1 with a-factor, treated with DMSO or 100 lM CPT, let in G 1 for 1h, and released into S phase. Three biological replicates have been performed. B, C Analysis of DNA content by flow cytometry of G 1 phase synchronized wild-type, rad3-102,rad3-102, rad51Δ,rad3-102 mus81,and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G 1 with a-factor, untreated, or irradiated with 20 J/m 2 UV-C, let in G 1 for 2h, and released into S phase. Three biological replicates have been performed. Data information: Asterisks indicate the progression of cells in S phase. 6of 19 EMBO reports 21:e49367 |2020 ª2020 The Authors EMBO reports Benjamin Pardo et al A BC Figure 4. Rad52 and Rad51 are required for HR-mediated replication fork protection. A Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52Δ,rad51Δ, mus81Δ,and pol32Δcells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 lM CPT for 2h and then pulse-labeled with 50 lM EdU for 20 min. DNA fibers were combed on silanized coverslips, and EdU- labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann–Whitney unpaired t-test, **P=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA. B Schematic representation of replication intermediates visualized by 2D gels. C Analysis of replication intermediates by 2D-gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81Δ,and rad3-102 mus81Δcells. Cells were synchronized in G 1 with a-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed. Data information: Accumulation of recombination molecules is indicated by white arrows. ª2020 The Authors EMBO reports 21:e49367 |2020 7of 19 Benjamin Pardo et al EMBO reports mus81Δcells, in which replication fork progression assayed by DNA combing was not more affected than in single mutants (Fig EV3B). Overall, our results show that CPT-mediated Top1 poisoning induces a global replication stress that requires HR factors for replication fork progression. However, Pol32 is not required to promote fork progression in response to CPT, suggesting that the strong reduction of nascent DNA tracks in rad52Δand rad51Δmutants is not due to the inability of blocked forks to perform BIR-mediated DNA synthesis. These results indicate that Pol32-dependent BIR synthesis is not the main pathway promoting the progression of replication forks blocked by Top1ccs. Even though we do not exclude that HR may promote the restart of blocked forks by BIR, we propose that HR is primarily required to protect blocked forks from degradation in response to Top1 poisoning. Mus81 resolves S phase-induced recombination events in G 2 /M Our data indicate that Mus81 is required to cope with the replication stress induced by Top1 poisoning but it does not promote replication progression in S phase. In human cells, Mus81 has been proposed to promote fork restart by cleaving stalled or reversed replication forks [54]. To understand this apparent contradiction and define the precise window of Mus81 activity, we assessed replication progression by two-dimensional (2D) neutral–neutral gel electrophoresis in synchronized cultures after release from G 1 phase, which was previously used to characterize the replication defects in rad3-102 cells. We studied the early replication origin ARS305 and the passively replicated region C besides it [82]. Notably, rad3-102 cells accumulated complex branched structures at region C (Fig 4B and C, see arrows), described as recombination or fork reversal events [65]. This assay gave us the opportunity to study the role of Mus81 in replication fork progression and recombination intermediate resolution in the rad3-102 background. As previously described, firing at ARS305 occurs slightly earlier in rad3-102 cells compared to wild type [65]. In the absence of Mus81 (mus81Δand rad3-102 mus81Δmutants), slower replication was observed around the ARS305, as the Y arc signal was still clearly observable at 60 min, while it had already disappeared in wild-type and rad3-102 cells (Fig 4C, left panel). This is consistent with a slower replication fork progression in mus81Δthan in wild-type cells observed by DNA combing in the absence of CPT-induced DNA damage (Figs 4A and EV3B). Complex branched structures did not accumulate in region C in the absence of Mus81 in rad3-102 mus81Δcells (Fig 4C, see arrows and Fig EV3D), suggesting that Mus81 does not process these substrates, observed in rad3-102 cells during S phase. More interestingly, we noted that, in a 100-min time-window after G 1 release, ARS305 fired twice in wild-type, mus81Δ,and rad3-102 cells, implying two rounds of replication. This was not observed in the rad3-102 mus81Δmutant (Fig 4C, left panel). As for ARS305 region, replication forks progressed only once through region C in rad3-102 mus81Δcells (Fig 4C, right panel). These results made us consider a cell cycle delay that could stem from a replication termination defect in the absence of Mus81 in rad3-102 cells. To explore this, we monitored the appearance of Rad52-YFP foci in cells exposed to CPT (Fig 5A). Wild-type cells were synchronized in G 1 with a-factor, incubated with CPT for 30 min, and then released from G 1 into S phase in the presence of CPT. Wild-type cells incubated with DMSO showed the appearance of a low amount of Rad52 foci in S phase 40 and 60 min after release from G 1 (Fig 5C). These foci disappeared when cells reached G 2 / M (Fig 5B and C), consistent with the observation that spontaneous Rad52 foci predominantly form in S phase cells [83]. When wild-type cells were released from the G 1 arrest in the presence of CPT, they accumulated six times more Rad52 foci than in control cells only when cells entered S phase at 40 min (Fig 5B and C). This accumulation continuously increased until cells reached the G 2 /M phase at 80 min and then started to decrease in later time points (Fig 5B and C). These results show that S phase entry is required for the initiation of recombination events induced by CPT, and these events are resolved after the completion of DNA replication in G 2 /M. When assessing the contribution of Mus81, we made two main observations. First, the increased accumulation of Rad52 foci during S phase caused by CPT exposure was not suppressed in the absence of Mus81 (Fig 5B and C), suggesting that Mus81 is not required to generate the substrates for recombination. Second, we observed that CPT-induced Rad52 foci in mus81Δcells accumulated over the entire time course experiment until 150 min, not showing the decrease observed in wild-type cells during the G 2 /M phase (Fig 5B and C). This phenotype was again confirmed in rad3-102 mus81Δcells, which accumulated more Rad52 foci than either single mutant ▸ Figure 5. Mus81 is involved in the processing of recombination intermediates in G 2 /M after replication restart. A Analysis of nuclear Rad52-YFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-YFP; red, mCHERRY-Pus1(nuclear compartment marker). Scale bar = 5lm. B, C Kinetic analysis of Rad52 foci formation. Wild-type and mus81Δcells containing Rad52-YFP and mCHERRY-Pus1were synchronized in G 1 with a-factor, treated with DMSO or 100 lM CPT, let in G 1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed. D Mms4-Flag10 phosphorylation analyzed by immunoblot in wild-type cells exposed to CPT. Wild-type cells were synchronized in G 1 with a-factor, treated with DMSO or 50 lM CPT, let in G 1 for 1h, and released into S phase. Cells were collected at the indicated time points and Mms4was immunodetected with anti-Flag antibodies. Clb2immunodetection serves as a marker for G 2 /M phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed. E CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G 2 /M-MUS81 alleles compared to the wild-type, mus81,mms4Δand cdc5-2mutants. Three biological replicates have been performed. F UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G 2 /M-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed. Source data are available online for this figure. 8of 19 EMBO reports 21:e49367 |2020 ª2020 The Authors EMBO reports Benjamin Pardo et al A C D E F B Figure 5. ª2020 The Authors EMBO reports 21:e49367 |2020 9of 19 Benjamin Pardo et al EMBO reports non-parametric test comparing unpaired groups of values was applied, for no Gaussian distributions were assumed. A P-value of <0.02 was deemed significant. Mean SEM are displayed for the concerned experiments, and sample size was not predetermined using any statistical method. Data availability Raw data that support the findings of this study others than the source data associated to this article (e.g., microscopy images) have not been deposited in a public database and are available from the corresponding authors upon reasonable request. Expanded View for this article is available online. Acknowledgements We thank L.S. Symington, J.H. Petrini, S.C. West, W.D. Heyer, J.A. Tercero, D. Branzei, S Piatti, H. Hombauer, and R. Kolodner for yeast strains and reagents. We thank Etienne Schwob and the DNA combing facility of Montpellier for providing silanized coverslips. We acknowledge the imaging and flow cytometry facility MRI, a member of the national infrastructure France-BioImaging supported by the “Agence Nationale pour la Recherche”(ANR-10-INBS-04, Investissements d’avenir), France. This work was funded by grants from the “Agence Nationale pour la Recherche”(ANR, France), the “Institut National du Cancer”(INCa, France), the “Ligue contre le Cancer”(équipe labellisée, France), and the MSDAvenir Fund, France, to PP and from the Spanish Ministry of Economy and Competitiveness (BFU2013-42918P, and Consolider Ingenio 2010 CSD2007-015), the “Junta de Andalucia”(BIO-1238 and CVI-4567, Spain), and the European Union (FEDER) to AA. BP was supported by fellowships from the “Fondation Recherche Médicale”(SPF20121226243, France) and from the Spanish Ministry of Economy and Competitiveness (JCI 2009-04101). MMC was recipient of a predoctoral FPU training grant from the Spanish Ministry of Economy and Competitiveness and a postdoctoral grant from the Labex EpiGenMed, an “Investissements d’Avenir”program (ANR-10-LABX-12-01), France. Author contributions Conceived and designed the experiments: BP, MM-C, AA and PP. Performed the experiments: BP, MM-C and TV. 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