Laurent Maloisel
email: laurent.maloisel@cea.fr

Emilie Ma

Jamie Phipps

Alice Deshayes

Stefano Mattarocci

Stéphane Marcand

Karine Dubrana

Eric Coïc
email: eric.coic@cea.fr

Ste ´phane Marcand

Rad51 filaments assembled in the absence of the complex formed by the Rad51 paralogs Rad55 and Rad57 are outcompeted by translesion DNA polymerases on UV-induced ssDNA gaps

come

Rad51 filaments assembled in the absence of the complex formed by the Rad51 paralogs Rad55 and Rad57 are outcompeted by translesion DNA polymerases on UV-induced ssDNA gaps

Introduction

In all living organisms, genomic DNA undergoes chemical modifications or crosslinking with proteins. These damages greatly compromise DNA replication because they induce replication fork stalling. DNA damage tolerance mechanisms have evolved to ensure completion of genome replication [START_REF] Branzei | DNA damage tolerance[END_REF], and rely on two main mechanisms: i) translesion synthesis (TLS) DNA polymerases and ii) the use of a homologous template, typically the newly synthesized sister chromatid. Specialized TLS polymerases efficiently insert nucleotides opposite and beyond lesions on DNA templates that block the replicative DNA polymerases [START_REF] Zhao | Translesion synthesis: Insights into the selection and switching of DNA polymerases[END_REF]. They possibly extend blocked primer/template junctions at replication forks or at single-stranded DNA (ssDNA) gaps left behind the forks [START_REF] Lehmann | Gaps and forks in DNA replication: Rediscovering old models[END_REF][START_REF] Hedglin | Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle[END_REF]. TLS polymerases are intrinsically error-prone and constitute a major source of DNA damage-induced mutagenesis [START_REF] Waters | Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance[END_REF][START_REF] Makarova | A four-subunit DNA polymerase ζ complex containing Pol δ accessory subunits is essential for PCNA-mediated mutagenesis[END_REF]. Recombination-mediated pathways, such as the template switching and homologous recombination (HR) pathways, mediate damage bypass through the annealing of the damaged ssDNA gaps to the intact homologous template on the sister chromatid [START_REF] Liberi | Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase[END_REF][START_REF] Branzei | SUMOylation regulates Rad18-mediated template switch[END_REF][START_REF] Branzei | DNA damage tolerance by recombination: Molecular pathways and DNA structures[END_REF]. HR, also referred to as the salvage pathway, can use homologous chromosomes as intact donors rather than sister chromatids [START_REF] Carr | Replication stress-induced genome instability: The dark side of replication maintenance by homologous recombination[END_REF]. As TLS, template switching and HR compete for the same ssDNA substrates, they can partly compensate for each other [START_REF] Lopes | Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions[END_REF][START_REF] Ma | Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells[END_REF][START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF]. However, template switching and HR are error-free lesion bypass mechanisms and counterbalance mutagenesis induced by TLS [START_REF] Fujii | Chronological switch from translesion synthesis to homology-Dependent gap repair in vivo[END_REF][START_REF] Masłowska | iDamage: a method to integrate modified DNA into the yeast genome[END_REF]. Thus, it is crucial to precisely characterize the factors that influence pathway choice to better understand genetic stability at replication forks.

The template switching pathway involves ubiquitin ligase and the Rad18/Rad6 pathway conjugating activities. In budding yeast, Rad6 and Rad18 induce PCNA mono-ubiquitination at its conserved K164 residue, whereas Rad5 and Mms2-Ubc13-dependent activities trigger its poly-ubiquitination at K164 [START_REF] Hoege | RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO[END_REF][START_REF] Torres-Ramos | Requirement of RAD5 and MMS2 for Postreplication Repair of UV-Damaged DNA in Saccharomyces cerevisiae[END_REF] and the formation of X-shaped intermediates between sister chromatids when replication is challenged by DNA damages [START_REF] Branzei | SUMOylation regulates Rad18-mediated template switch[END_REF]. Interestingly, the formation of these X-shaped intermediates relies also on the HR factors Rad51 and Rad52 [START_REF] Liberi | Rad51-dependent DNA structures accumulate at damaged replication forks in sgs1 mutants defective in the yeast ortholog of BLM RecQ helicase[END_REF][START_REF] Vanoli | Replication and recombination factors contributing to recombination-dependent bypass of DNA lesions by template switch[END_REF].

The HR pathway relies on the recombinase Rad51 that oligomerizes on ssDNA to form a right-handed helical nucleoprotein filament [START_REF] Conway | Crystal structure of a Rad51 filament[END_REF][START_REF] Short | High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy[END_REF]. This filament performs homology search and catalyzes DNA joint formation between ssDNA and double-stranded DNA (dsDNA) homologous partners, thereby leading to strand exchange [START_REF] Filippo | Mechanism of eukaryotic homologous recombination[END_REF][START_REF] Bell | Mechanics and Single-Molecule Interrogation of DNA Recombination[END_REF][START_REF] Haber | DNA Repair: The Search for Homology[END_REF]. Eventually, repair DNA synthesis occurs from the damaged DNA invading ends on undamaged template homologous DNA sequences [START_REF] Mehta | Sources of DNA double-strand breaks and models of recombinational DNA repair[END_REF]. Rad51 loading on RPA-coated ssDNA is a crucial step in HR and is mediated mainly by Rad52 in Saccharomyces cerevisiae and BRCA2 in metazoans [START_REF] Sung | Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase[END_REF][26][START_REF] Carver | Rad51 filament dynamics and its antagonistic modulators[END_REF]. Rad51 filament assembly also requires the activity of Rad51 paralogs (Rad55-Rad57 complex in S. cerevisiae; RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 in human cells; and the SHU complex in both [START_REF] Bonilla | RAD51 Gene Family Structure and Function[END_REF]). Rad55 and Rad57 share 20% identity with the RecA/Rad51 ATPase core region [START_REF] Kans | Nucleotide sequence of the RAD57 gene of Saccharomyces cerevisiae[END_REF][START_REF] Lovett | Sequence of the RAD55 gene of Saccharomyces cerevisiae: similarity of RAD55 to prokaryotic RecA and other RecA-like proteins[END_REF]. However, they do not form filaments on ssDNA, and they do not exhibit strand exchange activity [START_REF] Sung | Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase[END_REF][START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF]. Electron microscopy images showed the association of gold-labeled Rad55 with Rad51 filaments assembled on ssDNA (Rad51-ssDNA) [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF], and two-hybrid experiments revealed the interaction between Rad51 and Rad55 [START_REF] Hays | Complex formation in yeast double-strand break repair: Participation of Rad51, Rad52, Rad55, and Rad57 proteins[END_REF][START_REF] Johnson | Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57[END_REF]. However, recent single-molecule studies suggest that the interaction between the Rad55-Rad57 complex and Rad51 filaments is transient and the Rad55-Rad57 complex dissociates during filament extension [START_REF] Roy | The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination[END_REF]. As similar findings were obtained in nematodes [START_REF] Belan | Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins[END_REF], it has been proposed that RAD51 paralogs behave as classical chaperones to temporarily assist Rad51 filament formation. More studies are required to clearly describe the precise role of each complex of Rad51 paralogs. In addition, each complex might play a specific role depending on the initial HRinducing DNA lesion [START_REF] Garcin | Differential requirements for the RAD51 paralogs in genome repair and maintenance in human cells[END_REF].

Rad55-Rad57 role in DNA double strand break (DSB) repair is considered accessory on the basis of the weaker sensitivity of rad55 and rad57 mutants to ionizing radiation and to sitedirected DSBs compared with rad51 mutants [START_REF] Mozlin | Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination[END_REF][START_REF] Fung | Suppression of the double-strand-break-repair defect of the Saccharomyces cerevisiae rad57 mutant[END_REF]. Interestingly, this sensitivity seems to depend on the Srs2 helicase activity because it is partially suppressed by SRS2 ablation [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF][START_REF] Fung | Suppression of the double-strand-break-repair defect of the Saccharomyces cerevisiae rad57 mutant[END_REF].

In vitro experiments have shown that the Srs2 translocase activity disrupts Rad51 filaments [START_REF] Veaute | The Srs2 helicase prevents recombination by disrupting Rad51 nucleoprotein filaments[END_REF][START_REF] Krejci | DNA helicase Srs2 disrupts the Rad51 presynaptic filament[END_REF] and that the Rad55-Rad57 complex counteracts Srs2 to maintain Rad51 filaments on ssDNA [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF][START_REF] Roy | The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination[END_REF]. Therefore, it has been proposed that Rad51 filament assembly and disassembly, which are mediated by the Rad55-Rad57 complex and Srs2 respectively, provide a regulatory mechanism to control HR initiation. However, SRS2 deletion does not rescue spontaneous HR defects between direct repeats in the rad55 and rad57 mutants, indicating that the Rad55-Rad57 complex also acts independently of Srs2 [START_REF] Fung | Suppression of the double-strand-break-repair defect of the Saccharomyces cerevisiae rad57 mutant[END_REF].

To explain the different defects observed between spontaneous and DSB-induced HR, it was hypothesized that spontaneous HR between direct repeats is initiated by ssDNA gaps rather than DSBs [START_REF] Fung | Suppression of the double-strand-break-repair defect of the Saccharomyces cerevisiae rad57 mutant[END_REF]. Thus, the Rad55-Rad57 complex would play a more prominent role in HR when the initiating lesion is a ssDNA gap. In agreement, it has been observed that in cells lacking the TLS polymerases Pol z, RAD55 ablation leads to a synergistic increase in DNA damage sensitivity [START_REF] Xu | The yeast Shu complex utilizes homologous recombination machinery for error-free lesion bypass via physical interaction with a Rad51 paralogue[END_REF][START_REF] Rattray | The roles of REV3 and RAD57 in double-strand-breakrepair-induced mutagenesis of Saccharomyces cerevisiae[END_REF][START_REF] Ball | The yeast Shu complex couples error-free post-replication repair to homologous recombination[END_REF].

Here, we explored Rad55-Rad57 role in Rad51 filament formation on ssDNA gaps and in the balance between HR and TLS in S. cerevisiae. For that purpose, we induced interhomolog HR in diploid strains by ionizing radiation (IR) or UV radiation. IR generates DSBs and ssDNA gaps [START_REF] Cadet | Oxidatively generated complex DNA damage: Tandem and clustered lesions[END_REF], whereas UV generates mostly ssDNA gaps. UV-irradiated S. cerevisiae cells uncouple leading and lagging strand replication at irreparable UV lesions, thus generating long ssDNA regions on one side of the fork. Furthermore, small ssDNA gaps accumulate along replicated duplexes, likely resulting from repriming events downstream of the lesions on both leading and lagging strands. Translesion synthesis and homologous recombination counteract gap accumulation, without affecting fork progression [START_REF] Lopes | Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions[END_REF]. Recently, RPA foci were used as a read-out of ssDNA gaps forming upon DNA replication through Methyl-methane sulfonate (MMS)-and UV-induced DNA damage [START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF]. They predominantly form far away from sites of ongoing replication, and they do not overlap with any of the repair centers associated with collapsed replication forks or DNA double-strand breaks. Instead, they represent sites of post-replicative DNA damage bypass involving translesion synthesis and homologous recombination. Therefore, we also used this mark to evaluate the cross-talk between TLS and HR.

We found that the Rad55-Rad57 complex is essential for UV-induced HR, but only accessory for IR-induced HR. Interestingly, this essential role is mainly Srs2-independent, and UV-induced HR in Rad55-Rad57-deficient cells can be restored by inactivation of a TLS polymerase (Polz or Polη). Conversely, UV-induced HR cannot be restored upon inactivation of the template switch pathway in MMS2 deficient cells. Overall, our results show that the Rad55-Rad57 complex is essential for Rad51 filament assembly on UV-induced ssDNA gaps. When this complex is absent, Rad51 filaments cannot prevent the recruitment of TLS polymerases and counterbalance mutagenesis.

Results

The Rad55-Rad57 heterodimer is essential for UV-induced homologous recombination

To investigate Rad55-Rad57 role in HR, we analyzed UV-and IR-induced interhomolog HR in wild type (WT) and rad55Δ isogenic diploid strains. The previous observation that rad51 and rad52 mutant cells are very sensitive to IR indicates that IR generates DSBs, whereas these mutants are much more resistant to UV radiation, indicating that DSBs are probably rarely induced [START_REF] Chanet | Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase[END_REF][START_REF] Game | A genetic study of X-ray sensitive mutants in yeast[END_REF]. Additionally, genetic evidence suggests that UV-induced HR is triggered by ssDNA gaps [START_REF] Lettier | The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae[END_REF]. We measured interhomolog HR using two mutant alleles of ARG4: arg4-RV, a 2-bp deletion that ablates the EcoRV site at position +258, and arg4-Bg, a 4-bp insertion by fill-in of a BglII site at position +1,274 [START_REF] Coïc | Evidence for short-patch mismatch repair in Saccharomyces cerevisiae[END_REF] (Fig 1A). These alleles do not revert [START_REF] Coïc | Evidence for short-patch mismatch repair in Saccharomyces cerevisiae[END_REF] and only recombination in heteroallelic arg4-RV/arg4-Bg diploid cells results in the formation of a WT ARG4 gene primarily by non-reciprocal transfer covering one mutation [START_REF] Petes | Recombination in Yeast[END_REF].

The rad55Δ diploid strain was not sensitive to UV radiation (Fig 1B), but remarkably, UV-induced recombinant [Arg + ] frequency was strongly reduced in the rad55Δ diploid strain compared with WT cells (10-fold at 120 J/m 2 ) (Fig 1C). These results are very similar to those previously obtained with rad51Δ mutants [START_REF] Chanet | Semidominant mutations in the yeast Rad51 protein and their relationships with the Srs2 helicase[END_REF], suggesting that the Rad55-Rad57 complex is determinant in UV-induced HR. Conversely, rad55Δ diploid cells were sensitive to IR (Fig 1D), but γ-ray-induced HR frequencies were identical in rad55Δ and WT cells at high doses (400 Gy and 600 Gy) (Fig 1E). We observed the same phenotypes in the rad57Δ mutant (S1A-S1D Fig) . Likewise, it was previously reported that spontaneous HR rates are identical in rad57Δ and WT cells [START_REF] Mozlin | Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination[END_REF][START_REF] Friis | The effect of the mating-type alleles on intragenic recombination in yeast[END_REF], a result we confirmed here in rad55Δ cells (Fig 1F). These observations suggest that the Rad55-Rad57 complex plays a specific and essential role in UV-induced HR, probably at ssDNA gaps. This complex is also involved in the repair of IRinduced DSBs, as shown by the lower survival of rad55Δ mutant. However, it is not essential since the IR-induced HR frequencies are comparable to WT.

Resolution of UV-induced RPA foci is delayed in the rad55Δ mutant

To further examine the role of Rad55 in the management of ssDNA gaps induced by UV exposure, we monitored ssDNA by fluorescence microscopy using YFP-tagged Rfa1, the large subunit of the RPA complex [START_REF] Lisby | Choreography of the DNA damage response: Spatiotemporal relationships among checkpoint and repair proteins[END_REF]. It was indeed recently established that RPA foci formed upon UV-irradiation represent sites of post-replicative DNA damage bypass involving TLS and HR [START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF]. Exponentially growing cells were α-factor arrested in G1 and subjected or not to UV irradiation. In absence of irradiation, RPA foci were barely detectable in WT G1-arrested cells (S1E Fig). S1F). This illustrates the efficient post-replicative DNA damage bypass in WT cells.

Surprisingly, a steady level of 20% of rad55Δ G1-arrested cells exhibits RPA foci (S1E Fig). This rate slowly increases after UV-exposure to reach 60% of G1-arrested cells at 240 min (S1E Fig) . This newly described phenotype of rad55Δ cells is probably representative of an involvement of Rad55-Rad57 in the stabilization of repair intermediates of spontaneous and UV-induced DNA lesions in G1-arrested cells. 60 min after UV-exposure and release in the cell cycle, 80% of rad55Δ cells exhibit RPA foci (Fig 1H). Moreover, 31% of rad55Δ cells display three or more UV-induced RPA foci while this class is observed only in 10% of WT cells. In addition, contrary to WT, only 33% of RPA foci have been resolved 240 min post-irradiation. Finally, we observed an accumulation of rad55Δ cells in the second S phase 240 min after release from G1 arrest, probably related to the persistence of UV-induced gaps in absence of efficient HR (S1F Fig) . Altogether, these results confirm that Rad55-Rad57 is important to manage ssDNA gaps forming upon UV-exposure.

In order to confirm that the different behavior of Rad55-Rad57 after UV-and IR-exposure is related to the difference in the lesions induced, we also observed RPA foci formation in WT and rad55Δ cells exposed to IR. To allow haploid cells to repair γ-induced DSBs by HR between sister chromatids, cells were irradiated 60 min after their release from the G1 arrest (S2A Fig) . In WT and rad55Δ cells, we found the same proportion of cells displaying RPA foci after γ-rays exposure compared with UV, these foci being resolved with the same kinetics (S2B Fig). However, as already described in [START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF], 60 min after irradiation, 75% of the γ-irradiated cells with RPA foci display only one focus (S2B Fig), while this class represents 59% of UV-irradiated cells with RPA foci (Fig 1G). This difference could be attributed to the formation of a DSB repair center after γ-rays exposure while ssDNA gaps are not gathered after UVirradiation. This difference is even more pronounced in the rad55Δ mutant. Indeed, 76% of γirradiated cells with RPA foci show only one focus while this class with a single focus represents 44% of UV-irradiated cells with RPA foci. These differences support our conclusion that Rad51 filament are assembled mainly on ssDNA gaps after UV exposure, while some events might be gathered in repair centers after γ-rays irradiation.

UV-induced DNA lesions are channeled towards the REV3 error-prone DNA repair pathway in the rad55Δ mutant

Although UV-induced HR was almost abolished in rad55Δ cells, these cells were resistant to UV radiation. We hypothesized that in rad55Δ mutant cells, UV-induced ssDNA gaps at replication forks could be repaired by TLS instead of HR. The TLS polymerase Polz is required to bypass UV-induced DNA lesions, as illustrated by the strong UV-sensitivity of haploid cells lacking the Rev3 catalytic subunit of Polz (Fig 2A) [START_REF] Lemontt | Pathways of ultraviolet mutability in Saccharomyces cerevisiae. I. Some properties of double mutants involving uvs9 and rev[END_REF]. Conversely, rev3Δ diploid cells were much more resistant to UV radiation (Fig 2B), possibly due to HR promoted by MAT heterozygosity [START_REF] Friis | The effect of the mating-type alleles on intragenic recombination in yeast[END_REF][START_REF] Lovett | Characterization of null mutants of the RAD55 gene of Saccharomyces cerevisiae: effects of temperature, osmotic strength and mating type[END_REF][START_REF] Heude | a/α-control of DNA repair in the yeast Saccharomyces cerevisiae: Genetic and physiological aspects[END_REF][START_REF] Valencia-Burton | Different mating-type-regulated genes affect the DNA repair defects of saccharomyces RAD51, RAD52 and RAD55 mutants[END_REF]. In addition, Polz, is responsible for almost all UV-induced mutagenesis in yeast cells [START_REF] Lemontt | Pathways of ultraviolet mutability in Saccharomyces cerevisiae. I. Some properties of double mutants involving uvs9 and rev[END_REF][START_REF] Quah | The origin of spontaneous mutation in Saccharomyces cerevisiae[END_REF], as shown by the absence of UV-induced mutagenesis observed in the Polz essential component rev3 mutant [START_REF] Lemontt | Pathways of ultraviolet mutability in Saccharomyces cerevisiae. I. Some properties of double mutants involving uvs9 and rev[END_REF]. Therefore, rad55Δ cells should strongly rely on Polz and on its catalytic subunit Rev3 for UV resistance, and that UV-induced mutagenesis should increase in rad55Δ cells, as previously shown for spontaneous mutagenesis [START_REF] Xu | The yeast Shu complex utilizes homologous recombination machinery for error-free lesion bypass via physical interaction with a Rad51 paralogue[END_REF]. In agreement with previous studies, we confirmed that UV sensitivity is much higher in the rev3Δ rad55Δ double mutant than in the rev3Δ single mutant in haploid cells [START_REF] Xu | The yeast Shu complex utilizes homologous recombination machinery for error-free lesion bypass via physical interaction with a Rad51 paralogue[END_REF][START_REF] Rattray | The roles of REV3 and RAD57 in double-strand-breakrepair-induced mutagenesis of Saccharomyces cerevisiae[END_REF][START_REF] Ball | The yeast Shu complex couples error-free post-replication repair to homologous recombination[END_REF] (Fig 2A ) and also in diploid cells (Fig 2B). This UV sensitivity is comparable with those we previously observed in the rev3Δ rad51Δ double mutant [START_REF] Tellier-Lebegue | The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ[END_REF], confirming the essential role of Rad55-Rad57 in UVinduced HR. Therefore, the high resistance to UV of Rad55-deficient cells relies on the TLS pathway that could compensate for HR deficiency in these cells. Consequently, we expected an increase of mutagenesis frequency in rad55Δ cells. In agreement with this hypothesis, the frequency of UV-induced canavanine-resistant cells [START_REF] Whelan | The CAN1 locus of Saccharomyces cerevisiae: Fine-structure analysis and forward mutation rates[END_REF] was two-fold higher in the rad55Δ mutant than in WT cells (Fig 2C). This observation supports the channeling of DNA lesions towards the error-prone Polz-dependent pathway in rad55Δ cells.

Measurement of RPA foci in UV-irradiated cells confirmed that TLS can compensate for HR. While the number of RPA foci was about the same in the rev3Δ mutant and in the rad55Δ mutant (Figs 1G and2D), the rev3Δ rad55Δ double mutant shows RPA foci in almost all cells with 64% of this population displaying at least three foci (Fig 2D). We also observed that rev3Δ mutants display an accumulation of cells in the second S phase 240 min after release from G1 arrest, while rev3Δ rad55Δ double mutant shows a block in the first G2 phase after the release (S3 Fig) . The number of cells displaying RPA foci barely decreased with time in rev3Δ rad55Δ double mutant. However, the number of cells displaying at least three foci decreases strongly, which could represent repair events but also the disappearance of ssDNA gaps through degradation or breakage in the absence of repair. Considering the low survival of UV-irradiated rev3Δ rad55Δ cells, this more likely indicates that many ssDNA gaps formed during the first S phase after release cannot be repaired in absence of TLS and HR, leading to faulty mitosis. We also observed that the number of spontaneous RPA foci is 2-fold higher in the rad55Δ rev3Δ mutant compared with the rad55Δ mutant, indicating a large accumulation of ssDNA gaps

UV-induced DNA lesions are channeled to the HR pathway in cells lacking TLS polymerases

The finding that UV-induced DNA lesions can be channeled to the TLS pathway in rad55Δ cells suggested that such lesions could be managed by the HR pathway in TLS-deficient cells. In that case, TLS-deficient cells should display a hyper-recombinogenic phenotype. To test this hypothesis, we measured the frequencies of UV-induced HR in rev3Δ (Polz mutant) and rad30Δ (Polη mutant) cells. As expected, HR frequency was strongly increased in rev3Δ cells and to a lower extent in the rad30Δ mutant (Fig 3A and3B). However, UV sensitivity was similar in diploid cells harboring the rev3Δ or rad30Δ mutation (Figs 2B and3C). We propose that in the rad30Δ mutant, UV-induced DNA lesions can be bypassed by both HR and Polz, while Polη cannot always take over Polz function in rev3Δ cells, resulting in higher HR frequency. Additionally, it is important to note that accordingly with the strong sensitivity of rev3Δ cells exposed to UV, HR cannot compensate for all UV-induced DNA lesions that are supported by the TLS pathway.

Translesion DNA polymerases prevent UV-induced HR in the rad55Δ mutant

We showed that UV-induced HR is strongly decreased in Rad55-Rad57 deficient cells. We also observed that the TLS and HR pathways compete for the same UV-induced ssDNA gaps, because mutagenesis was increased in rad55Δ mutants, and HR in rev3Δ mutants. Therefore, the recruitment of TLS polymerases at the lesion site might decrease HR in rad55Δ cells. We measured HR frequencies and observed a significantly increased frequency in the rev3Δ rad55Δ double mutant compared with rad55Δ cells (Fig 4A). This indicated that Polz effectively impairs HR in the absence of the Rad55-Rad57 complex. We propose that the Rad55-Rad57 complex is important to stabilize Rad51 filaments on ssDNA gaps at the DNA lesion site, thus limiting the recruitment of TLS polymerases. Our results also suggest that in the absence of the Rad55-Rad57 complex, Rad51 filaments are functional enough to promote some HR events (Fig 4A), although this is not sufficient to improve UV resistance (Fig 2). We observed the same results when a Polη mutation (rad30Δ) was combined with rad55Δ Next, we asked whether UV-induced HR can be observed independently of the Rad55-Rad57 complex in cells deficient for the template switching pathway. Therefore, we measured the cell survival and recombination frequencies after UV radiation of cells in which MMS2 was deleted. We found that the rad55Δ mms2Δ double mutant was more sensitive than the single mutants (Fig 4E), suggesting that some template switching events can occur independently of HR. Strikingly, the frequency of UV-induced recombinants was as low in the rad55Δ mms2Δ double mutant as in the rad55Δ mutant (Fig 4F). This suggests that the template switching pathway does not compete with HR between homologous chromosome and that TLS polymerases still prevent HR in the rad55Δ mms2Δ double mutant. On the other hand, the recombination frequency in the mms2Δ single mutant was increased, but only after exposure to the highest UV dose (120 J/m 2 , Fig 4F ).

The acute UV sensitivity of the rad55Δ rev3Δ double mutant is partially suppressed by SRS2 deletion

As the Rad55-Rad57 complex limits Rad51 filament destabilization by Srs2 at DSBs [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF][START_REF] Roy | The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination[END_REF], we asked whether Srs2 would be involved in the defective UV-induced HR associated with rad55Δ or rad57Δ. To address this question, we measured UV-induced HR frequencies in rad55Δ srs2Δ and rad57Δ srs2Δ double mutants. We found that they were similar to those observed in rad55Δ and rad57Δ single mutants (Figs 5A and5B, S4A, andS4B). However, rev3Δ rad55Δ srs2Δ diploid cells were less sensitive to UV radiation than rev3Δ rad55Δ cells (Fig 5C). Thus, the Rad55-Rad57 complex is also involved in the protection of Rad51 filaments against Srs2 at ssDNA gaps formed after UV radiation, but this can only be seen in the absence of Polz. When Polz is active, the HR rate is probably too low in rad55Δ mutant cells to allow the detection of the effect of Rad51 displacement by Srs2.

Additionally, the level of resistance conferred by the deletion of SRS2 in rad55Δ rev3Δ cells do not reach those of the rev3Δ single mutant (Fig 5C). This indicates that the Rad55-Rad57 complex plays a Srs2-independent role in UV-induced HR that is only revealed in rev3Δ mutants. We propose that the Rad55-Rad57 complex provides stability to Rad51 filaments independently of the protection against Srs2.

To sustain our conclusion, we analyzed UV-induced RPA foci in srs2Δ cells. The resolution of UV-induced RPA foci is delayed in srs2Δ cells compare to WT but in a lesser extent than in rad55Δ cells (Figs 1G and5E). Moreover, the delay in UV-induced RPA foci resolution observed in the rad55Δ single mutant (Figs 1G andS1F) is the same than in the rad55Δ srs2Δ double mutant (Figs 5F andS5), suggesting that the antirecombinase activity of Srs2 does not account for the delay observed in the rad55Δ mutant. Similarly, the number of spontaneous and UV-induced RPA foci observed in rev3Δ rad55Δ srs2Δ haploid cells was the same as the number observed in the rev3Δ rad55Δ double mutant (Fig 5F). Moreover, the absence of Srs2 does not suppress the block in the first G2 phase after the release observed in the rev3Δ rad55Δ double mutant following UV irradiation (S3 and S5 Figs).

The Rad55-Rad57 complex is essential for the formation of UV-induced lethal recombination events

We confirmed that the srs2Δ diploid strain is very sensitive to UV radiation as reported before [START_REF] Aboussekhra | RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene[END_REF]; however, this sensitivity was completely suppressed in rad55Δ srs2Δ and rad57Δ srs2Δ mutant cells (Figs 5D andS4C). Previous genetic studies led to the concept of toxic Rad51-dependent recombination intermediates that accumulate in the absence of Srs2 [START_REF] Gangloff | Homologous recombination is responsible for cell death in the absence of the Sgs1 and Srs2 helicases[END_REF][START_REF] Klein | Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Δ with other DNA repair genes in Saccharomycescerevisiae[END_REF][START_REF] Fabre | Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication[END_REF]. Therefore, on the basis of the complete suppression of srs2Δ-associated UV sensitivity by rad55Δ and rad57Δ, we propose that the Rad55-Rad57 complex participates in the formation of UVinduced Rad51 filaments that are toxic for the cells in the absence of Srs2. In agreement with previous reports [START_REF] Aboussekhra | RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene[END_REF][START_REF] Rong | The hyper-gene conversion hpr5-1 mutation of Saccharomyces cerevisiae is an allele of the SRS2/RADH gene[END_REF][START_REF] Esta | Rad52 sumoylation prevents the toxicity of unproductive Rad51 filaments independently of the anti-recombinase Srs2[END_REF], we observed a strong UV-induced hyper-recombination phenotype in Srs2-deficient cells. This phenotype was completely suppressed by the concomitant deletion of Rad55 or Rad57 (Figs 5A andS4A).

The translesion DNA polymerases Polz and Polη are not essential for DSB repair

The large impact of the rad55Δ rev3Δ double mutant on cell survival after UV radiation suggested that the Rad55-Rad57 complex and Polz play an important and specific role in UVinduced DNA repair. Although the Rad55-Rad57 complex is not essential for DSB repair [START_REF] Fung | Suppression of the double-strand-break-repair defect of the Saccharomyces cerevisiae rad57 mutant[END_REF], we wanted to determine the potential role of TLS polymerases in DSB repair in rad55Δ cells. To this aim, we measured the repair of a DSB induced at the MAT locus upon expression of the HO endonuclease controlled by a galactose-inducible promoter. The DSB was repaired by HR using an ectopic MATa-inc sequence inserted in chromosome V [START_REF] Esta | Rad52 sumoylation prevents the toxicity of unproductive Rad51 filaments independently of the anti-recombinase Srs2[END_REF][START_REF] Ira | Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast[END_REF] (Fig 6A). After DSB induction, survival was decreased by 3-fold in rad55Δ cells compared with WT cells (Fig 6B). Conversely, survival of the rev3Δ, rad30Δ, and rev3Δ rad30Δ mutants was not affected by DSB induction, and they did not change the sensitivity of the rad55Δ mutant. This indicates that the TLS polymerases are not required for DSB repair efficiency.

Discussion

The Rad55-Rad57 complex is essential for HR-mediated repair of UVinduced ssDNA gaps Genetic studies provided evidence that ssDNA gaps are the major initiator of spontaneous and UV-induced HR in yeast [START_REF] Game | A genetic study of X-ray sensitive mutants in yeast[END_REF][START_REF] Quah | The origin of spontaneous mutation in Saccharomyces cerevisiae[END_REF]. In addition, electron microscopy and two-dimensional gel electrophoresis showed that UV-irradiated cells accumulate ssDNA gaps, likely resulting from re-priming events downstream of stalled replication forks at UV-induced DNA lesions [START_REF] Lopes | Multiple mechanisms control chromosome integrity after replication fork uncoupling and restart at irreparable UV lesions[END_REF]. UV-induced ssDNA gaps were also inferred from a physical assay [START_REF] Ma | Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells[END_REF] and from the study of RPA foci distribution relatively to sites of ongoing replication [START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF]. Interestingly, both studies reported that TLS and HR counteract ssDNA gap accumulation.

Here, we show that the complex formed by the Rad51 paralogs Rad55 and Rad57 is essential for UV-induced ssDNA gaps repair, while accessory at DSB sites. We observed a strong decrease of HR frequencies in the rad55Δ and rad57Δ mutants specifically after UV radiation. Moreover, we confirmed the synergistic increase of UV sensitivity upon depletion of both Rad55 and Polz in haploid and diploid cells (Fig 2A and2B). This was confirmed by the large increase in the number of UV-induced RPA foci observed in the rad55Δ rev3Δ double mutant (Fig 2D). We did not observe this negative interaction after induction of a site-directed DSB (Fig 6). In addition, we found that UV-induced mutagenesis at the CAN1 locus was increased in the rad55Δ mutant (Fig 2C), and that UV-induced HR was increased in Pol z and Pol η deficient cells (Fig 3A and3B). Most importantly, we observed UV-induced HR in the absence of the Rad55-Rad57 complex only in cells deficient for TLS (Fig 4). These data suggest that the Rad55-Rad57 complex might stabilize Rad51 filaments on ssDNA gaps to prevent the recruitment of the TLS polymerases.

The Rad55-Rad57 complex plays three prominent roles in UV-induced ΗR One of the best defined role of Rad55-Rad57 is the protection of Rad51 filaments from Srs2 at DSBs [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF][START_REF] Roy | The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination[END_REF]. Here, we found that although the defect in UV-induced HR observed in rad55Δ cells was not suppressed by SRS2 deletion (Fig 5A and5B), the very high UV sensitivity of the rad55Δ rev3Δ double mutant was partially suppressed by srs2Δ (Fig 5C). This suggests that the Rad55-Rad57 complex protects Rad51 filaments against Srs2 also at UV-induced ssDNA gaps, although Esc2-and Elg1-dependent mechanisms also regulate negatively Srs2 at sites of stalled replication forks [START_REF] Urulangodi | Local regulation of the Srs2 helicase by the SUMO-like domain protein Esc2 promotes recombination at sites of stalled replication[END_REF][START_REF] Arbel | Access to pcna by srs2 and elg1 controls the choice between alternative repair pathways in saccharomyces cerevisiae[END_REF]. Additional studies are required to determine the interplay between these regulating factors.

The increased UV-sensitivity of the rev3Δ rad55Δ srs2Δ triple mutant compared with the rev3Δ single mutant (Fig 5C ) clearly indicates that the Rad55-Rad57 complex plays a Srs2-independent role in UV-induced HR. We propose that the second role of this complex is to provide stability to Rad51 filaments, independently of the protection against Srs2. In agreement with this conclusion, we found that in rad55Δ or rad57Δ mutants, srs2Δ-associated UV-sensitivity was completely suppressed (Fig 5D andS4C). This indicates that in the absence of Srs2 activity, Rad51 stabilization by the Rad55-Rad57 complex would lead to lethal events, possibly initiated by ssDNA gaps that could block replication fork restart. On the basis of the results of single-molecule studies, it was proposed that the Rad55-Rad57 complex helps Rad51 filament formation by acting as a chaperone [START_REF] Roy | The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination[END_REF], in agreement with our observations. Remarkably, UV-induced HR frequency increased in rad55Δ cells upon Polz or Polη deletion (Fig 4). Therefore, the third role of the Rad55-Rad57 complex would be to allow HR to outcompete the TLS polymerases. Rad51 filament stabilization by the Rad55-Rad57 complex on ssDNA at the lesion site could prevent the recruitment of TLS polymerases by PCNA because of structural constraints. In the absence of Rad55-Rad57, unstable Rad51 filaments cannot prevent the loading of the TLS polymerases that inhibit HR and induce mutagenesis. TLS polymerase depletion in rad55Δ mutants would allow some HR events to occur, but the inherent instability of Rad51 filaments would make them rare and explain the low survival rate of rev3Δ rad55Δ mutant cells. Our results clearly highlight that UV-induced lesions can be channeled from HR to TLS and vice versa, when one pathway is inactivated. This provides further support to a model in which HR and TLS can share common substrates [START_REF] Ma | Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells[END_REF][START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF].

Model for the activity of the Rad55-Rad57 complex in ssDNA gap repair

Our genetic data suggest three different and essential functions for the Rad55-Rad57 complex in UV-induced interhomolog HR initiated at ssDNA gaps (Fig 7A). First, this complex is required for Rad51 filament formation and stabilization that are essential for efficient strand exchange at ssDNA gaps. The 5'ends of DSBs are resected to generate 3' ssDNA that can invade the homologous donor. Conversely, 3' ssDNA is not directly available at ssDNA gaps. Therefore, the Rad55-Rad57 complex may be required to form Rad51 filaments that can invade without 3' ssDNA extremities. Alternatively, the Rad55-Rad57 complex can be involved

PLOS GENETICS

in the 3' end denaturation at the lesion site to generate a 3' ended ssDNA by recruitment of a DNA helicase. Second, the Rad55-Rad57 complex within Rad51 filaments counterbalances Srs2 activity through a mechanism that remains poorly understood [START_REF] Liu | Rad51 paralogues Rad55-Rad57 balance the antirecombinase Srs2 in Rad51 filament formation[END_REF][START_REF] Roy | The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination[END_REF]. Third, the Rad55-Rad57 complex could inhibit TLS polymerases by limiting their loading at the primer/template junction within ssDNA gaps, probably through the stabilization of Rad51 filaments (Fig 7B -7E). This competition might be crucial to control mutagenesis resulting from gap-filling by Polz, but further studies will be required to understand how the access of HR and TLS are regulated at the site of lesions and to determine the nature of the lesions that can only be managed by TLS, as shown by the acute sensitivity of UV-irradiated rev3Δ mutant. Thus, in our model, Rad51 filaments formed with the Rad55-Rad57 complex preserve the genome stability through ssDNA gap repair by HR, but also through competition with TLS on common ssDNA gap substrates.

The interplay between HR and TLS in mammals

The interplay between error-prone and error-free lesion-bypass pathways is documented in E. coli and yeast (the present study, [START_REF] Wong | Processing of DNA Polymerase-Blocking Lesions during Genome Replication Is Spatially and Temporally Segregated from Replication Forks[END_REF][START_REF] Fujii | Chronological switch from translesion synthesis to homology-Dependent gap repair in vivo[END_REF]), and it has been recently investigated in mammals. First of all, it was recently shown that BRCA-deficient tumors sensibility to cisplatin and synthetic lethality with PARP inhibitors were associated with a BRCA default in preventing replication-associated gaps rather than in preventing DSBs [START_REF] Panzarino | Replication Gaps Underlie BRCA Deficiency and Therapy Response[END_REF][START_REF] Cong | Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency[END_REF]. More specifically, HR can be triggered by PRIMPOL-dependent ssDNA gaps behind stalled replication forks [START_REF] Piberger | PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts[END_REF]. Moreover, BRCA1-and BRCA2-defective breast cancer cells, display an increased somatic mutational load specific of TLS polymerases [START_REF] Alexandrov | Signatures of mutational processes in human cancer[END_REF][START_REF] Nik-Zainal | Landscape of somatic mutations in 560 breast cancer whole-genome sequences[END_REF][START_REF] Nolan | Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer[END_REF][START_REF] Wen | Association of BRCA1-And BRCA2-deficiency with mutation burden, expression of PD-L1/ PD-1, immune infiltrates, and T cell-inflamed signature in breast cancer[END_REF]. Accordingly, a recent study found that BRCA1/2-defective cancer cells rely much more on TLS for repair of PRIMPOL-dependent ssDNA gaps [START_REF] Taglialatela | REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps[END_REF]. In addition, similarly to PARP1 or DNA polymerase Polθ inhibitors that are used to treat HR-deficient cancers [START_REF] Hurley | In the Clinic[END_REF][START_REF] Zhou | A first-in-class polymerase theta inhibitor selectively targets homologous-recombination-deficient tumors[END_REF][START_REF] Zatreanu | Polθ inhibitors elicit BRCAgene synthetic lethality and target PARP inhibitor resistance[END_REF], the small molecule JE-RH-06 disrupts the interaction between the TLS polymerases REV1 and Polz and shows preferential cytotoxicity in BRCA1-deficient cancer cells [START_REF] Taglialatela | REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps[END_REF][START_REF] Wojtaszek | A Small Molecule Targeting Mutagenic Translesion Synthesis Improves Chemotherapy[END_REF]. Thus, it would be interesting to determine whether the association of cancer-associated RAD51 paralog mutations and TLS mutations leads to a synergic sensitivity to replication fork blocking lesions, as we observed in yeast in the rad55Δ rev3Δ double mutant. If an acute sensitivity is found, one might expect a cytotoxicity associated with TLS polymerase inhibitors in RAD51 paralog-deficient cancer cells.

Materials and methods

Yeast strains, plasmids and media

The S. cerevisiae strains used in the present study are isogenic derivatives of FF18733 (his7-2, leu2-3,112, lys1-1, trp1-289, ura3-52), JKM146 (hml::ADE1 MATalpha hmr::ADE1, arg5,6:: MATa-inc::HPH1, ade3::GAL::HO) and W303 (ADE2 leu2-3,112 his3-11,15 trp1-1, ura3-1) and are listed in S1 Table . Yeast cells were incubated at 30˚C for all the experiments described. Gene deletions were performed by PCR-mediated one-step replacement [START_REF] Baudin | A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae[END_REF][START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF]. Mutants were selected on YPD medium containing 300 mg/L geneticin (Sigma) or nourseothricin (clo-NAT; Werner BioAgents). All deletions were confirmed by PCR amplification of genomic DNA. All media were prepared as previously described [START_REF] Sherman | Getting started with yeast[END_REF].

Irradiation, cell survival assay, and measurement of DNA damage-induced HR frequency

Cells in stationary phase and in exponential growth phase were used for irradiation with UV and γ-rays, respectively. UV irradiation was performed using a 264 nm source. Yeast cells were irradiated with 30, 60, 90 and 120 J/m 2 (2 J/m 2 /s dose rate). γ-irradiation was performed using a GSR D1 irradiator (Gamma-Service Medical GmbH). It is a self-shielded irradiator with four sources of 137 Cesium. The total activity was 180.28 TBq in March 2014. As yeast cells resuspended in 1 mL of sterile H 2 O were irradiated in 1.5ml plastic microtubes, dosimetry was performed using plastic microtubes with a cylindrical ionizing chamber 31010 (PTW, Freiburg, Germany) following the American Association of Physicists in Medicine recommendations [START_REF] Ma | AAPM protocol for 40-300 kV x-ray beam dosimetry in radiotherapy and radiobiology[END_REF]. This ionizing chamber has a cavity of 0.125 cm 3 calibrated with 137 Cesium in air kerma free in air and the reference number of our facility is 210382401. The polarity and the ion recombination were measured for this 137 Cesium source. Each measurement was corrected with the KTP factor to take into account the variations in temperature and atmospheric pressure. Yeast cells were irradiated at 100, 200, 400 and 600 Gy (single doses) and with a 12 Gy/ min dose rate that takes the radioactive decrease into account.

Before (UV) or after (γ-rays) irradiation, cells were plated at the appropriate dilution on rich medium (YPD) to measure the survival rate, and on synthetic plates without arginine to quantify the number of HR events between ARG4 heteroalleles. HR frequencies were determined by dividing the number of recombinant colonies growing on selective medium by the number of unselected colonies subjected to the same dose of irradiation. The values obtained were corrected by subtracting the number of recombinants on the non-irradiated plates. The mean percentage from at least three independent experiments is presented.

Measurement of spontaneous heteroallelic HR

Rates of spontaneous HR between two heteroalleles of ARG4 were determined by fluctuation tests using the method of the median [START_REF] Spell | Determination of mitotic recombination rates by fluctuation analysis in Saccharomyces cerevisiae[END_REF]. The reported rates were obtained from three independent experiments, each performed with nine independent 2-ml cultures started with less than 200 cells and incubated at 30˚C for three days. The significance of the rates is indicated by the 95% confidence interval [START_REF] Spell | Determination of mitotic recombination rates by fluctuation analysis in Saccharomyces cerevisiae[END_REF].

Measurement of UV-induced mutagenesis

UV-induced mutagenesis was measured with the CAN1 forward-mutation assay [START_REF] Whelan | The CAN1 locus of Saccharomyces cerevisiae: Fine-structure analysis and forward mutation rates[END_REF]. UVinduced mutagenesis frequencies were obtained by dividing the number of colonies growing on synthetic plates without arginine and containing l-canavanine (Sigma, 30 mg/l) (i.e., canavanine-resistant, Can R , cells) by the number of cells that survived irradiation counted on rich media YPD. The number of Can R colonies obtained after irradiation was corrected by subtracting the number of spontaneous Can R colonies growing on non-irradiated plates.

Cell growth, synchronization and irradiation before microscopy

Cells were grown at 30˚C in synthetic complete medium (Formedium). Before UV-and γ-irradiation, growing cells were synchronized in G1 using 17 mg/ml α-factor for 180 min. They were released from the G1 arrest by three consecutive washes with sterile H 2 O. Cells were resuspended in 50 ml of sterile H 2 O and UV-irradiated in a glass plate of diameter 190 mm. UV irradiation was performed using a 264 nm source. Yeast cells in water were irradiated with 60 J/m2 (2 J/m2/s dose rate) then resuspended in fresh complete medium. γ-irradiation was performed as described for survival studies. Cells were then resuspended in fresh complete medium. Time-course experiment were started with yeast cultures adjusted to OD 600 0.5.

Microscopy

Live-cell images were acquired using a wide-field inverted microscope (Leica DMI-6000B) equipped with Adaptive Focus Control to eliminate Z drift, a 100×/1.4 NA immersion objective with a Prior NanoScanZ Nanopositioning Piezo Z Stage System, a CMOS camera (ORCA-Flash4.0; Hamamatsu) and a solid-state light source (SpectraX, Lumencore). The system is piloted by MetaMorph software (Molecular Device).

YFP images were acquired at indicated time points after alpha factor block release and UV or γ-rays irradiation; 19 focal steps of 0.20 μm were acquired with an exposure time of 100 ms using a solid-state 500-nm diode and a YFP filter (excitation 470-510 nm and dichroic 495 nm; Chroma Technology Corp.). A single bright-field image on one focal plane was acquired at each time point with an exposure of 50ms. All the images shown are z projections of z-stack images. Image analysis was achieved following processing with ImageJ software (National Institutes of Health), using scripts written in ImageJ macro language. Bright-field and maximum intensity projections of YFP images were merged, Rfa1-YPD foci were then quantified in 2D.

Measure of DNA content by flow cytometry

1 ml of cells were resuspended in 70% Ethanol and kept at 4˚C. Cells were centrifuged for 3 min at 7,000 RPM and washed once in PBS, resuspended in PBS with RNase A (0.5 mg/ml) for 2 hours at 50˚C. Cells were centrifugated and resuspended in PBS with iodure propidium (50 μg/ml) for one hour at room temperature. Cells were diluted 10-fold in PBS and cell aggregates were dissociated by sonication. DNA content analysis was performed using LSRII flow cytometer (BD Biosciences) and analyzed with the FlowJo software.

Survival following DSB formation

Cells were grown overnight in liquid culture medium containing lactate acid instead of glucose. Survival following HO-induced DSB was measured as the number of cells growing on galactose-containing medium divided by the number of colonies growing on YPD. The results shown are the mean values of at least 3 independent experiments.

  After UV-exposure, only a few G1-arrested WT cells displayed RPA foci (3.2% and 12.5% after 60 min and 240 min post-irradiation, respectively; S1E Fig). Unirradiated WT cells released into the cell cycle also exhibit few RPA foci (10% of the cells 60 min after release; Fig 1H). In marked contrast, 58% of WT irradiated cells displayed at least one RPA foci 60

Fig 1 .

 1 Fig 1. Rad55 plays a major role in UV-induced HR. (A) Schematic representation of the genetic system used. Frameshift mutations were introduced at the EcoRV site (RV, ±2 bp) or the BglII site (Bg, +4 bp). HR between the two ARG4 mutant alleles can restore a WT ARG4 allele associated with the [Arg+] phenotype. (B) Survival and (C) [Arg+] recombinant frequencies after UV exposure of WT and rad55Δ diploid cells. (D) Survival and (E) [Arg+] recombinant frequencies after γ irradiation of WT and rad55Δ diploid cells. (F) Spontaneous mitotic [Arg+] recombinant rates ([Arg+] x 10 -7 /cell/generation) of WT and rad55Δ diploid strains. (G) Experimental scheme for microscopy analysis of RPA foci: Cells arrested in G1 phase with alpha-factor (αF) were exposed to UV before release into the cell cycle. Samples were collected for analysis every hour for four hours. Representative images of Rfa1-YFP cells are presented. Scale bars are 2 μm. (H) Quantification of Rfa1-YFP foci detected by fluorescence microscopy in WT and rad55Δ cells exposed or not to UV. Error bars indicate SDs from three independent experiments. https://doi.org/10.1371/journal.pgen.1010639.g001

Fig 2 .

 2 Fig 2. Channeling UV-induced DNA lesions into the REV3-dependent pathway in rad55Δ cells. (A) Survival of haploid cells following UV radiation. (B) Survival of diploid cells following UV radiation. (C) UV-induced mutagenesis in WT and rad55Δ cells determined by quantifying the Can R mutant frequency for the indicated UV doses. (D) Quantification of Rfa1-YFP foci detected by fluorescence microscopy in rev3Δ and rad55Δ rev3Δ cells not irradiated or after UV exposure. Error bars indicate SDs from three independent experiments. https://doi.org/10.1371/journal.pgen.1010639.g002

Fig 3 .

 3 Fig 3. Channeling UV-induced DNA lesions into the HR pathway in TLS-deficient cells. (A) UV-induced [Arg+] recombinant frequencies in WT and rev3Δ diploid cells. (B) UV-induced [Arg+] recombinant frequencies in WT and rad30Δ diploid cells. (C) Survival of diploid rad30Δ cells after UV radiation. https://doi.org/10.1371/journal.pgen.1010639.g003

Fig 4 .

 4 Fig 4. RAD55-independent UV-induced HR in TLS-deficient cells. (A) UV-induced [Arg+] recombinant frequencies in rad55Δ rev3Δ diploid cells. The very strong UV sensitivity displayed by the double mutant precluded the quantification of the UV-induced recombinant rate at UV doses higher than 40J/m 2 . (B) Survival of rad55Δ rad30Δ diploid cells following UV radiation. (C) UV-induced [Arg+] recombinant frequencies in rad55Δ and rad30Δ diploids cells. (D) Close-up view of (C) to exclude the hyper-recombinogenic phenotype of rad30Δ. (E) Rad55Δ mms2Δ diploid cell-survival upon UV radiation. (F) UV-induced [Arg+] recombinant frequencies in rad55Δ and mms2Δ diploid cells. https://doi.org/10.1371/journal.pgen.1010639.g004

Fig 5 .

 5 Fig 5. Srs2 role in the rad55Δ mutant exposed to UV radiation. (A) UV-induced [Arg+] recombinant frequencies in rad55Δ srs2Δ diploid cells. (B) Close-up view of (A) to exclude the hyper-recombinogenic phenotype of srs2Δ. (C) The acute UV sensitivity displayed by the rad55Δ rev3Δ mutant is partially suppressed by SRS2 deletion. The red arrow between the rev3Δ rad55Δ and rev3Δ rad55Δ srs2Δ survival curves highlights the partial srs2Δ suppression. (D) Survival curves of diploid cells following UV radiation. The acute sensitivity to UV radiation of the diploid srs2Δ strain is suppressed by rad55Δ (blue arrow). (E) Quantification of Rfa1-YFP foci detected by fluorescence microscopy in srs2Δ cells not irradiated or after UV exposure. (F) Quantification of Rfa1-YFP foci detected by fluorescence microscopy in rad55Δ srs2Δ and rad55Δ rev3Δ srs2Δ cells not irradiated or after UV exposure. Error bars indicate SDs from three independent experiments. https://doi.org/10.1371/journal.pgen.1010639.g005

Fig 6 .

 6 Fig 6. TLS DNA polymerases are not required for cell survival after a site-specific DSB. (A) Schematic representation of the system used to measure repair of a HO-induced DSB by gene conversion between ectopic copies of MAT. (B) Cell viability after DSB formation. https://doi.org/10.1371/journal.pgen.1010639.g006

Fig 7 .

 7 Fig 7. Model of Rad55-Rad57 roles in the repair of ssDNA gaps by HR behind replication forks. The figure shows an ssDNA gap generated after stalling of a replicative DNA polymerase at a UV-induced DNA lesion. This gap is a substrate for both the HR and TLS pathways. (A) The three roles of the Rad55-Rad57 complex in Rad51 filament formation on ssDNA gaps are highlighted: (1) formation of functional Rad51 filaments; (2) protection against Srs2 dismantling activity; (3) prevention of TLS polymerase recruitment at the primer/template junction. (B) In WT cells, Rad55-Rad57 heterodimers form and stabilize Rad51 filaments on ssDNA leading to HR, avoiding bypass by TLS. Rad51 filaments could prevent the recruitment of the TLS polymerases Polz and Polη (collectively named TLS Pol) by covering the 3' extremity blocked at the DNA lesion. (C) In the rad55Δ mutant, inefficient and/or fewer Rad51 filaments are formed that cannot prevent the recruitment of the TLS polymerases. Therefore, more UV-induced DNA lesions are channeled towards the TLS pathway (bold arrow). (D) In the rev3Δ or in the rad30Δ mutant, the absence of Polz or Polη allows channeling UV-induced DNA lesions towards the HR pathway (bold arrow). (E) In the rad55Δ rev3Δ and rad55Δ rad30Δ double mutants both the HR and the TLS pathways are impaired. https://doi.org/10.1371/journal.pgen.1010639.g007

PLOS Genetics | https://doi.org/10.1371/journal.pgen.1010639February 7, 2023

Acknowledgments

We thank Jim Haber and Pablo Radicella, for critical and careful reading of the manuscript. We thank Ve ´ronique Me ´nard from the radiation facility of our institute for her help with the use of the GSRD1 irradiator. We also appreciate the help of Elisabetta Andermarcher with the English editing.

This research was funded by Electricite de France (EDF), Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA) Radiobiology call, Tandem Call CEA-PIC3i Institut Curie in Radiobiologie (to EC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the paper and its Supporting Information files.

Supporting information


Charbel Rahhal
email: charbel.rahal@loria.fr

Hala Skaf-Molli

Pascal Molli
email: molli@loria.fr

SWOOKI: A Peer-to-peer Semantic Wiki

In this paper, we propose to combine the advantages of semantic wikis and P2P wikis in order to design a peer-to-peer semantic wiki. The main challenge is how to merge wiki pages that embed semantic annotations. Merging algorithms used in P2P wiki systems have been designed for linear text and not for semantic data. In this paper, we evaluate two optimistic replication algorithms to build a P2P semantic wiki.

Introduction

Nowadays, Wikis are the most popular web-based collaborative writing tools. In spite of their popularity, wikis suffer from the difficulty of navigation and information retrieval. To overcome these problems, some traditional wiki systems turned into semantic wikis. Semantic Wiki is a wiki engine with technologies from semantic web1 to embed formalized knowledge, content, structures and links in wiki pages. Popular semantic wikis are based on the client-server architecture. All wiki pages reside on a single server that controls operations of distributed users. Consequently, scalability, performance, fault-tolerance and load balancing are major challenges for current semantic wikis. In addition, centralized architecture suffers from censorship problem and does not support off-line work. An approach to solve these problems is to shift from centralized architecture to full distributed (peer to peer) one. In this paper, we address the challenge of transforming a P2P wiki system into a P2P semantic wiki system. In fact, merging algorithms used in P2P wiki systems have been designed for linear text and not for semantic data. In this paper, we show how we can use the existent optimistic replication algorithms to build a P2P semantic wiki.

SWOOKI Approach

SWooki is the first attempt to build a peer to peer semantic wiki. SWooki is based on Wooki [START_REF] Weiss | Wooki: a p2p wiki-based collaborative writing tool[END_REF] a peer-to-peer wiki system. SWooki integrates the semantic web technology by following the philosophy of Semantic Media Wiki [START_REF] Völkel | Semantic Wikipedia[END_REF]. The semantic annotations are embedded in the wiki text via a wiki markup e.g. typed links. It follows the use of wikis for ontologies approach. A formal ontology emerges during the edition of the wiki pages. SWooki provides the same functionalities of any server-based semantic wiki. In addition, SWooki allows the following three interesting use cases for P2P wikis: (1) a massive collaboration, (2) the offline editing, and (3) an ad-hoc collaboration [START_REF] Androutsellis-Theotokis | A survey of peer-to-peer content distribution technologies[END_REF]. In order to combine P2P wiki system with semantic wiki systems, it is very important to know how semantic wikis represent their semantic data and how they combine textual parts with semantic parts. The main issue that we address is how to merge wiki pages that contain semantic annotations and if this combination changes the behavior of the Semantic Media Wiki. In this paper, we investigate how we can combine the Wooki with the Semantic Media wiki. We called this combination SWooki. We adopt Wooki [START_REF] Weiss | Wooki: a p2p wiki-based collaborative writing tool[END_REF] because it supports all use cases for a P2P wiki system previously cited. In Wooki, wiki pages are replicated over all members of the p2p overlay network. A wiki page is considered as a sequence of lines. Each server hosts a copy of pages and can autonomously offer the wiki service. Page copies at each site are maintained by an optimistic replication mechanism called Woot [START_REF] Oster | Data consistency for P2P collaborative editing[END_REF] that disseminates changes and ensures consistency. Woot ensures the CSCW principles of convergence and user intentions. The only two available merging algorithms for peer-to-peer wikis are the Thomas rule [START_REF] Johnson | RFC0677: Maintenance of duplicate databases[END_REF] and Woot strategy. These algorithms handle linear text. The merge using these algorithms is done by the server which differs from the merge done by the users during concurrent editing in centralized semantic wikis. In case of Thomas rule, only the modifications of the user that lastly saved are kept. In case of Woot, the result includes the modifications of all users. However, the result in Woot is produced by the server and must be reviewed by a human in order to verify its accuracy. The Woot algorithm preserves users intentions, all concurrent effects are visible in the final version of the wiki page. If the user wants to change the result of the merge, he can do that easily. In conclusion, Swooki adopts the Woot algorithm because it provides the best solution for the merge by keeping all concurrent changes made by the users without any lost of updates. The SWooki approach allows to build a P2P semantic wiki very easily by integrating semantic annotations into a P2P wiki. It allows also to balance the load of queries. It provides a cheap way to have many replicas of the same semantic wiki. This total replication of semantic data can be used to distribute semantic queries on different replicas.

www.w3.org/2001/sw