Julia Godau

Lorenza P Ferretti

Anika Trenner

Emeline Dubois

Christine Von Aesch

Antoine Marmignon

Lauriane Simon

Aurélie Kapusta

Raphaël Guérois

Mireille Bétermier
email: mireille.betermier@i2bc.paris-saclay.fr

Christine Von Aesch

Alessandro A Sartori
email: sartori@imcr.uzh.ch

Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia

Keywords: Paramecium tetraurelia, CtIP, Meiosis, DNA double-strand breaks, Homologous recombination, DNA end resection

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Introduction

DNA double-strand breaks (DSBs) are the most lethal type of DNA damage, and, if incorrectly repaired, can drive tumorigenesis [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. On the other hand, induction of DSBs by ionizing radiation (IR) or DNA topoisomerase poisons like camptothecin (CPT) represents a common therapeutic strategy to effectively eliminate cancer cells [START_REF] Pommier | Roles of eukaryotic topoisomerases in transcription, replication and genomic stability[END_REF]. Cells have evolved two major pathways for the repair of DSBs: Non-homologous end-joining (NHEJ), which ligates broken ends without the need of extensive processing, and homologous recombination (HR), which requires an intact homologous DNA template for repair [START_REF] Chang | Non-homologous DNA end joining and alternative pathways to double-strand break repair[END_REF]4]. The first step of HR is termed DNA-end resection and involves nucleolytic processing of DSB ends to generate 3' single-stranded DNA (ssDNA) overhangs that are immediately covered by the replication protein A (RPA). RPA subsequently gets replaced by the Rad51 recombinase, which initiates homology search and strand invasion [START_REF] Symington | Mechanism and regulation of DNA end resection in eukaryotes[END_REF]. In eukaryotes, DNA-end resection is initiated by the concerted action of the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) nuclease complex in association with Sae2/Ctp1/CtIP [START_REF] Symington | Mechanism and regulation of DNA end resection in eukaryotes[END_REF]. At the protein level, human CtIP and its counterparts in other species are predicted to be largely intrinsically disordered, with the exception of a conserved N-terminal coiled-coil domain that assembles into a tetrameric 'dimer-of-dimers' complex [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF][START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF]. In addition, a short C-terminal stretch showing the highest degree of amino acid sequence similarity within CtIP protein members is commonly referred to as the 'Sae2-like' domain [START_REF] Andres | CtIP/Ctp1/Sae2, molecular form fit for function[END_REF]. Human CtIP and its functional counterparts in S. pombe (Ctp1), A. thaliana and C. elegans have been identified as homologs of S. cerevisiae Sae2 (Sporulation in the absence of Spo eleven 2), a protein that is required for the initiation of DNA-end resection in meiotic and mitotic yeast cells [START_REF] Penkner | A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination[END_REF][START_REF] Uanschou | A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene[END_REF][START_REF] Limbo | Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control doublestrand break repair by homologous recombination[END_REF][12][13][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF].

The biochemical properties of recombinant Sae2/Ctp1/CtIP proteins have been extensively studied in recent years, significantly contributing to our mechanistic understanding of DNAend resection [START_REF] Andres | CtIP/Ctp1/Sae2, molecular form fit for function[END_REF]. First, both Sae2 and CtIP were shown to directly associate with the MRX and MRN complex, respectively, and to stimulate Mre11's weak endonuclease activity in cleaving the 5' strand [START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Cannavo | Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks[END_REF][START_REF] Paull | The 3" to 5" exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks[END_REF][START_REF] Paull | Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex[END_REF][START_REF] Anand | Phosphorylated CtIP Functions as a Cofactor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection[END_REF][START_REF] Garcia | Bidirectional resection of DNA doublestrand breaks by Mre11 and Exo1[END_REF][START_REF] Paudyal | Dna2 initiates resection at clean DNA double-strand breaks[END_REF]. In a second step, DNA is processed in 3' to 5' direction from the nick towards the DSB end by Mre11's intrinsic exonuclease activity, meanwhile 5' to 3' exonucleases such as Exo1 and Dna2 progressively resect the 5' DNA strand, ultimately generating a 3' single-stranded DNA (ssDNA) overhang compatible for Rad51 binding [START_REF] Mimitou | Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing[END_REF][START_REF] Zhu | Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends[END_REF][START_REF] Nicolette | Mre11-Rad50-Xrs2 and Sae2 promote 5' strand resection of DNA double-strand breaks[END_REF][START_REF] Niu | Mechanism of the ATPdependent DNA end-resection machinery from Saccharomyces cerevisiae[END_REF].

In the germline, the formation of programmed DSBs, generated by the topoisomeraselike Spo11 protein, is required for genetic recombination during meiosis [START_REF] Lam | Mechanism and regulation of meiotic recombination initiation[END_REF]. Throughout evolution, CtIP and the Mre11 nuclease complex are strictly required for meiotic recombination by removing covalently attached Spo11 from DSB ends and, thereby, allowing DNA-end resection to start [START_REF] Arora | Genetic separation of Sae2 nuclease activity from Mre11 nuclease functions in budding yeast[END_REF][START_REF] Ma | Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair[END_REF][START_REF] Lemmens | COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining[END_REF][START_REF] Hartsuiker | Ctp1CtIP and Rad32Mre11 nuclease activity are required for Rec12Spo11 removal, but Rec12Spo11 removal is dispensable for other MRN-dependent meiotic functions[END_REF][START_REF] Hartsuiker | Distinct Requirements for the Rad32Mre11 Nuclease and Ctp1CtIP in the Removal of Covalently Bound Topoisomerase I and II from DNA[END_REF]. Paramecium tetraurelia, like all ciliates, is a unicellular eukaryote that contains two functionally distinct types of nuclei: a large somatic macronucleus (MAC, 800n) responsible for gene expression, but not transmitted to sexual progeny, and two identical diploid germline micronuclei (MIC) that undergo meiosis during the sexual cycle [START_REF] Arnaiz | The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences[END_REF]. In P. tetraurelia, correct assembly of newly developing MACs requires the elimination of 25-30% of MIC DNA, including the precise excision of thousands of ~45,000 short internal eliminated sequences (IES) [START_REF] Arnaiz | The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences[END_REF]. DNA elimination is mediated through the sequential action of PiggyMac, a domesticated piggyBac transposase essential for cleaving DNA at IES ends, and the NHEJ pathway [START_REF] Arnaiz | The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences[END_REF][START_REF] Kapusta | Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV-Dependent End Joining[END_REF][START_REF] Baudry | PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF][START_REF] Dubois | Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements[END_REF][START_REF] Marmignon | Kumediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF]. With regards to recombinatorial repair, a recent inventory of meiosis-specific genes in ciliates revealed the presence of two gene copies each for PtMRE11 and PtCtIP, consistent with multiple whole-genome duplication events in the early stages of P. tetraurelia evolution [START_REF] Chi | Meiosis gene inventory of four ciliates reveals the prevalence of a synaptonemal complex-independent crossover pathway[END_REF][START_REF] Aury | Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia[END_REF]. Remarkably, with a length of only 198 amino acids, PtCtIP paralogs would constitute the smallest In the present study, we sought to characterize the function of PtCtIP in vivo and in vitro. Comparative sequence analysis reveals that PtCtIPa and PtCtIPb, collectively termed as PtCtIP, share the minimal domain architecture present in all Sae2/Ctp1/CtIP proteins. PtCtIP expression is required for the processing of Spo11-dependent meiotic DSBs, an essential prerequisite to the production of gametic nuclei and subsequent development of a new somatic MAC in sexual progeny. Purified recombinant PtCtIP binds with high affinity to double-stranded branched DNA molecules but lacks detectable intrinsic nuclease activity.

Moreover, PtCtIP-DNA interaction, but not PtCtIP-Mre11 interaction, strongly depends on a highly conserved 'RHR' motif located in the C-terminus. Finally, we provide evidence that the 'RHR' motif mediates efficient recruitment of human CtIP to sites of DNA damage ensuring efficient DSB resection and repair by HR.

Results

Paramecium tetraurelia encodes two protein copies with a domain organization shared by CtIP homologs

CtIP protein sequences are poorly conserved across the eukaryote phylogeny and vary greatly in their molecular size, particularly between yeast and mammalian homologs (Fig. 1A). Schizosaccharomyces pombe Ctp1 (294 amino acids, aa) represents the smallest CtIP homolog that has been characterized so far [START_REF] Limbo | Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control doublestrand break repair by homologous recombination[END_REF]. To screen for additional members of the Sae2/Ctp1/CtIP protein family in other species, we ran iterative PSI-BLAST searches using as query the amino acid sequence of the conserved C-terminal domain of human CtIP (75 aa). Interestingly, we retrieved two putative CtIP-like polypeptides in Paramecium tetraurelia comprised of only 198 aa sharing more than 80% sequence identity (Fig. 1A and Supplemental Fig. 1A). Closer examination of the P. tetraurelia predicted proteome indeed revealed the existence of two closely related CtIP paralogs, PtCtIPa and PtCtIPb (ParameciumDB accession numbers GSPATP00020534001 and GSPATP00027328001) [START_REF] Arnaiz | ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia[END_REF].

PtCtIPa and PtCtIPb gene copies resulted from the most recent whole genome duplication (WGD) that took place before the evolutionary split of the Paramecium aurelia genus into 15 sibling species including P. tetraurelia [START_REF] Aury | Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia[END_REF].

To gain further insight into the protein sequence and domain architecture of PtCtIP, we adopted a hierarchical alignment procedure allowing us to align different evolutionarily conserved regions of CtIP homologs. The N-terminus of PtCtIP features five repeating heptad

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sequences, which are predicted to assemble into a coiled-coil structure typically present in Sae2/Ctp1/CtIP proteins (Fig. 1A and Supplemental Fig. 1B). Moreover, upstream of the heptad repeats, a short α-helical region previously reported to mediate CtIP tetramerization can be distinguished with high confidence in PtCtIP (Supplemental Fig. 1B) [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF][START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF]. The phylogenetic tree built from a multiple sequence alignment restricted to the conserved Cterminal domain demonstrates that PtCtIP clusters together with protozoan sequences, as expected, while strong sequence divergences are primarily observed between ascomycetes, including S. cerevisiae Sae2 (Fig. 1B andC). Closer examination of the PtCtIP C-terminal region revealed the presence of two conserved, closely spaced, short linear sequence motifs:

'CxxC' and 'RHR' (Fig. 1C). The 'RHR' motif, located at aa positions 181-183 of PtCtIP, was shown to promote DNA-bridging activity of Ctp1 in vitro [START_REF] Andres | CtIP/Ctp1/Sae2, molecular form fit for function[END_REF]. We further noted that PtCtIP is the only family member lacking a C-terminal amino-acid extension after the cyclindependent kinase (CDK) consensus sequence motif (S/TP) required for DNA-end resection in yeast and human cells (Fig. 1C) [START_REF] Huertas | CDK targets Sae2 to control DNA-end resection and homologous recombination[END_REF][START_REF] Huertas | Human CtIP mediates cell cycle control of DNA end resection and double strand break repair[END_REF]. In fact, PtCtIP lacks any additional CDK phosphorylation sites reported to promote the interaction of human CtIP with Nbs1 FHA/BRCT domains [START_REF] Wang | The Interaction of CtIP and Nbs1 Connects CDK and ATM to Regulate HR-Mediated Double-Strand Break Repair[END_REF], suggesting that it is not prone to be regulated in a cell-cycle regulated manner but rather at the level of transcription. Consistent with this observation, no apparent homolog of Nbs1 exists in P. tetraurelia. In conclusion, our in silico analysis predicts that PtCtIP is mainly composed of the conserved N-and C-terminal regions of the Sae2/Ctp1/CtIP family, making it the smallest CtIP ortholog known to date.

Paramecium CtIP genes are required for the recovery of viable progeny during autogamy

Autogamy is a self-fertilization process encountered in the Paramecium aurelia group of species (Supplemental Fig. 2A). Upon starvation, mature P. tetraurelia cells that have undergone at least 20 vegetative fissions can start MIC meiosis in the absence of a sexual partner. A fully homozygous zygotic nucleus is formed through the fusion of two identical gametic nuclei. Following two successive nuclear divisions that take place in the absence of cell division, two mitotic copies of the zygotic nucleus give rise to the new MICs while new MACs differentiate from the other two copies. We surveyed the expression of PtCtIP compared to PtSPO11 in P. tetraurelia cells at different autogamy stages. Northern blot analysis revealed an early transcription induction peak during MIC meiosis for both PtCtIP genes simultaneously to PtSPO11 (Supplemental Fig. 2B). This result was confirmed by deep sequencing of mRNAs (Fig. 2A) [START_REF] Arnaiz | Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression[END_REF]. As expected from the conservation of the meiosis process in Paramecium, the genome of P. tetraurelia carries two recently duplicated PtMRE11 genes. Northern blot hybridization and deep sequencing of mRNAs revealed that PtMRE11 genes have quite different patterns of expression: PtMRE11a (ParameciumDB accession number GSPATG00020413001) exhibits a constitutively low level of expression at all stages of the sexual cycle, while PtMRE11b (ParameciumDB accession number GSPATG00023641001) is strongly induced during meiosis (Fig. 2A and Supplemental Fig. 2B) [START_REF] Arnaiz | ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia[END_REF]. Notably, PtMRE11b levels peak at the same time during meiosis as both PtCtIP and PtSPO11 mRNAs (Fig. 2A and Supplemental Fig. 2B).

To gain insight into the function of CtIP in P. tetraurelia, we used an RNAi strategy to silence the expression of PtCtIP genes during autogamy, individually or altogether. Because both genes are actively transcribed during meiosis (Fig. 2A and Supplemental Fig. 2B), vegetative growing cells were fed on dsRNA-producing bacteria and left to starve in the feeding medium until autogamy started. Quantitative analysis of mRNA levels using northern blot hybridization demonstrated an efficient RNAi-mediated downregulation of PtCtIPa and b (up to ~10 fold) relative to control RNAi (Fig. 2B and Supplemental Fig. 2C). During PtCtIP knockdowns, a large fraction of autogamous cells harboured only fragments from the old MAC, but did not develop new MACs (Supplemental Fig. 2C andD). No MICs could be detected in these cells either, suggesting that depletion of PtCtIP triggers an early defect during the sexual cycle and prevents the formation of a functional zygotic nucleus. At later stages, cells with two MICs and a single large nucleus surrounded by several smaller fragments started to appear in PtCtIP knockdowns (orange population in Supplemental Fig. 2C). These could result from the overgrowth of a minority of viable post-autogamous progeny that succeeded in making their new MACs, throughout the long incubation time of each experiment (3 to 4 days).

To check whether knockdown of PtCtIP or PtMRE11 genes has a debilitating effect on the ability of sexual progeny to resume vegetative growth, we measured the percentage of post-autogamous survivors with functional new MACs. Knockdown of individual PtCtIPa or b genes did not cause any reduction of viable progeny, suggesting that PtCtIP genes can compensate each other (Fig. 2C). However, the concomitant inactivation of both PtCtIPa and b expression resulted in ~70% lethality in sexual progeny (Fig. 2C). In contrast, silencing of PtSPO11 or PtMRE11b alone was sufficient to induce strong lethality in post-autogamous progeny, consistent with their respective meiosis-specific expression profiles (Fig. 2A and Supplemental Fig. 2B). These results indicate that, despite its short size, PtCtIP, like PtMre11, is fully functional and essential for DSB repair during meiosis in P. tetraurelia.

Abnormal meiosis and lack of new developing MACs in cells depleted for PtCtIP genes

The complete absence of new MICs and MACs in PtCtIP knockdowns likely reflects an early defect in the formation of a functional zygotic nucleus. In the ciliate T. thermophila, SAE2/COM1 knockout was shown to block the progression of MIC meiosis at the meiosis I stage, as a result of defective repair of meiotic DSBs and inefficient pairing of homologous chromosomes, eventually leading to chromosome "disintegration" and MIC degeneration [START_REF] Lukaszewicz | MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena[END_REF]. Because autogamy is poorly synchronous in P. tetraurelia, we switched to conjugation in order to synchronize MIC meiosis and zygotic nucleus formation in the population [START_REF] Bétermier | Timing of developmentally programmed excision and circularization of Paramecium internal eliminated sequences[END_REF].

We used anti-γ-tubulin antibodies to specifically label the MICs, the zygotic nucleus and its mitotic division products [START_REF] Klotz | Gamma-tubulin and MTOCs in Paramecium[END_REF] in conjugating cells co-silenced for both PtCtIP genes (Fig. 3A and Supplemental Fig. 3). This experiment confirmed that MIC meiosis proceeded normally at the cytological level, until the 4-MIC stage (i.e. meiosis I) in a large majority of cells.

Abnormal patterns started to appear at meiosis II, with partial or complete loss of MIC meiotic products and complete absence of a zygotic nucleus in a majority of mating pairs, while normal fragmentation of the old MAC was still observed, suggesting that no fragmentation checkpoint exists in P. tetraurelia. Therefore, CtIP depletion in P. tetraurelia does not allow the formation of a zygotic nucleus, because it triggers an arrest right after meiosis I.

In other organisms, the absence of CtIP also results in abortive meiosis, due to the accumulation of unrepaired Spo11-dependent DSBs. In S. pombe, ctp1 C-terminal mutations severely reduce the yield of viable spores [START_REF] Ma | Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair[END_REF]. In budding yeast, sae2∆ mutant cells do not form tetrads, but sporulation is restored in the sae2∆spo11∆ double mutant background, even though the resulting spores are not viable [12,13]. To get further insight into the meiotic function of the P. tetraurelia CtIP homologs, we knocked down PtSPO11 by inducing a ∆SPO11 somatic deletion (∆SPO11MAC) in strain 51 new (see Methods). We then submitted wild-type and ∆SPO11MAC cells to RNAi against PtCtIP genes and to a control RNAi against ND7. As expected, ∆SPO11MAC cells did not yield viable progeny in either condition (PtCtIP or control RNAi, Supplemental Fig. 4A). During the progression of autogamy, significant differences were observed between ∆SPO11MAC and wild-type cells (Fig. 3B). In wild-type cells, PtCtIP silencing strongly impaired the formation of developing new MACs, as already observed. In the ∆SPO11MAC mutant submitted to a control RNAi, new developing MACs were detected, although not quite with the same efficiency as in the wild-type background.

However, as indicated by the low survival rate in the progeny of ∆SPO11MAC cells (Supplemental Fig. 4A), these new MACs were not functional, suggesting that in P.

PtCtIP DNA-binding depends on a conserved C-terminal RHR motif

Our genetic analyses underscored an essential role for CtIP during autogamy and conjugation in P. tetraurelia. In order to investigate how PtCtIP contributes to DNA-end resection at the molecular level, we purified full-length recombinant PtCtIPa protein from insect cells (Supplemental Fig. 5A andB). First, we aimed to characterize its DNA-binding properties by in vitro gel shift assays using 5'-end radiolabeled 50-mer oligonucleotide substrates (Supplemental Table S1). At a fixed protein/DNA molar ratio, we observed that PtCtIPa specifically interacts with double-stranded, but not single-stranded nucleic acids (Fig. 4A). Further protein titration and DNA competition experiments revealed that PtCtIPa exhibits a slight binding preference for forked versus blunt-ended DNA (Fig. 4B and Supplemental Fig. 5C). We noted that PtCtIPa-DNA complexes migrated near the top of the gel, indicative of the formation of rather large multimeric assemblies, in particular when considering the low molecular weight of Paramecium CtIP (Fig. 4A andB). Interestingly, however, recent studies demonstrated that both Ctp1 and human CtIP N-terminal domains can form stable tetramers consisting of two coiled-coil dimers [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF][START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF]. Thus, given that both structural motifs mediating Ctp1/CtIP tetramerization are conserved in PtCtIP (Supplemental Fig. 1B), it is conceivable to predict that recombinant PtCtIPa also exists as a tetramer in solution resulting in the formation of high molecular weight complexes upon DNA binding.

Indeed, size exclusion chromatography analysis indicated a dominant oligomeric state for PtCtIPa and MBP-PtCtIPa, the latter of which eluting at similar retention volumes as Ctp1 (Supplemental Fig. 5D). Moreover, residual amounts of uncleaved MBP-PtCtIPa (67 kDa) fusion protein present in our preparations (Supplemental Fig. 5B) might explain why the DNA mobility shift is even slightly bigger compared with Ctp1 (Fig. 4B).

In addition to the N-terminal multimerization domain, Andres et al. reported a 'RHR' DNA-interaction motif located at the Ctp1 C-terminus [START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF]. As the 'RHR' motif is evolutionarily highly conserved among CtIP homologs from different species including P. tetraurelia (Fig. 1C), we expressed and purified recombinant PtCtIPa-AHA with R181 and R183 mutated to alanine residues (Fig. 4C and Supplemental Fig. 5B). Gel shift assays showed that the PtCtIP-AHA mutant was completely defective in binding to the forked-DNA CtIP proteins (Fig. 4D). Notably, employing the same radiolabeled forked-DNA substrates, we could not detect any DNA cleavage activity associated with recombinant PtCtIPa (Supplemental Fig. 6A andB).

Remarkably, MBP-pulldown assays revealed that both wild-type (wt) PtCtIPa and the PtCtIP-AHA mutant are able to interact with recombinant human Mre11-Rad50 (MR) complex, suggesting an evolutionarily conserved mode of interaction between the two factors (Supplemental Fig. 6C). Human CtIP was shown to stimulate Mre11 endonuclease activity in vitro using different DNA substrates [START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Anand | Phosphorylated CtIP Functions as a Cofactor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection[END_REF]. For instance, using closed-circular singlestranded PhiX174 DNA as a substrate, human CtIP specifically triggered robust MRdependent endonucleolytic cleavage in the presence of magnesium but only mildly stimulated MR in the presence of manganese [START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF]. Under the exact same assay conditions as described therein, we observed that PtCtIPa is enhancing MR-mediated cleavage of PhiX174 DNA to a similar extent as human CtIP (Supplemental Fig. 6D andE). Interestingly, we could not reveal any difference in the amount of DNA cleavage products between PtCtIPa-wt and PtCtIP-AHA mutant, indicating that PtCtIP's DNA binding ability is largely dispensable for functionally interacting with MR in vitro. Taken together, our combined genetic and biochemical analysis of PtCtIP strongly supports its key role in DSB repair-related processes. and immunofluorescence microscopy analysis revealed that the two cell lines display similar cell cycle profiles and GFP-CtIP nuclear localisation patterns (Supplemental Fig. 8A andB).

Moreover, CtIP-AHA mutant was efficiently phosphorylated at its nearby cyclin-dependent kinase (CDK) site (T847) critical for DNA-end resection [START_REF] Huertas | Human CtIP mediates cell cycle control of DNA end resection and double strand break repair[END_REF], suggesting that DNA binding is not a prerequisite for regulatory CtIP modifications (Supplemental Fig. 8C). Given that recombinant PtCtIP-AHA was defective in DNA binding, we first examined the assembly of GFP-CtIP at DNA lesions induced by laser micro-irradiation. Importantly, previous studies have shown that Sae2/CtIP and Mre11 are independently recruited to sites of DSBs [START_REF] Lisby | Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins[END_REF][START_REF] Chen | Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair[END_REF][START_REF] Eid | DNA end resection by CtIP and exonuclease 1 prevents genomic instability[END_REF].

Remarkably, we observed that the GFP-CtIP fluorescent intensity at DSB-containing tracks marked with γH2AX was significantly reduced in the AHA mutant compared with the wt, suggesting that DNA binding is a critical determinant for CtIP retention at damaged chromatin (Fig. 5A and Supplemental Fig. 8D). CtIP is required for CPT-induced hyperphosphorylation of RPA2 at serines 4/8, a surrogate marker for DSB resection revealed by western blotting (Fig. 5B, lanes 1-3) [START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Kousholt | CtIPdependent DNA resection is required for DNA damage checkpoint maintenance but not initiation[END_REF]. We observed that re-expression of CtIP-wt fully restored RPA2 hyperphosphorylation in CtIP-depleted cells, whereas the rescue with CtIP-AHA was only partial (Fig. 5B, lane 7). Of note, GFP-CtIP expression as well as ATM phosphorylation levels were comparable between the two cell lines, indicating proficient upstream DSB signalling (Fig. 5B). Consistently, using a flow cytometry-based approach to measure DNA-end resection [START_REF] Forment | A flow cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells[END_REF][START_REF] Ferretti | Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection[END_REF], we found that RPA retention on damaged chromatin is strongly reduced in AHA mutant compared to wt expressing cells (Fig. 5C). To further evaluate the impact of CtIP DNA binding on the processing of replication-associated DSBs formed at collapsed replication forks, we depleted endogenous CtIP from our U2OS clones

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and measured their viability following chronic exposure to CPT. In agreement with previous data [START_REF] Ferretti | Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection[END_REF], activation of CtIP-wt expression by Dox administration efficiently rescued CPT hypersensitivity of CtIP-depleted cells (Fig. 5D). By contrast, cells expressing the CtIP-AHA mutant remained sensitive to CPT, indicative of an HR defect (Fig. 5D). Finally, to directly measure HR efficiency, we made use of the traffic-light reporter (TLR) assay system [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF] and engineered U2OS-TLR clones stably expressing siRNA-resistant FLAG-tagged CtIP-wt and -AHA (Fig. 5E). Strikingly, whereas HR and DNA-end resection were largely restored in CtIP-depleted cells complemented with CtIP-wt, they were still significantly compromised in both CtIP-AHA expressing clones (Fig. 5E and Supplemental Fig. 8E), indicating that the DNA-binding ability of CtIP is a critical determinant of resection-dependent DSB repair by HR. Collectively, these findings establish that CtIP-DNA interaction via the 'RHR' motif

Discussion

PtCtIP represents a miniature ortholog of the CtIP protein family

Spontaneous or damage-induced DSBs need to be accurately repaired to maintain genome integrity and suppress tumorigenesis. During meiosis, however, hundreds of DBSs are deliberately induced by Spo11-dependent cleavage and subsequently repaired by homologous recombination to generate genetic diversity. In mammalian cells, faithful processing of both accidental and programmed DSBs requires the concerted action of the MRN complex and CtIP. Remarkably, while Mre11 and Rad50 are highly conserved and present throughout evolution, CtIP-related proteins have so far only been identified in eukaryotes [START_REF] Stracker | The MRE11 complex: starting from the ends[END_REF][START_REF] Lim | A novel mode of nuclease action is revealed by the bacterial Mre11/Rad50 complex[END_REF]. Moreover, CtIP homologs have rapidly diverged in primary sequence and size ranging from 294 aa (S. pombe Ctp1) to 897 aa (human CtIP). In this study, we have characterized the genetic and biochemical activities of P. tetraurelia CtIP (PtCtIP), with only 198 aa in size, representing the smallest member of the Sae2/Ctp1/CtIP protein family known to date. Our primary sequence analysis revealed that PtCtIP is basically composed of the two hallmark structural elements common to all CtIP proteins: an N-terminal coiled-coil domain critical for dimer/tetramer assembly and a highly conserved C-terminus harboring a short linear motif implicated in DNA binding (Fig. 1 and Supplemental Fig. 1). In contrast, the large, intrinsically disordered middle region in human CtIP, shown to coordinate multiple protein-protein interactions and to harbor nuclease motifs [START_REF] Andres | CtIP/Ctp1/Sae2, molecular form fit for function[END_REF][START_REF] Makharashvili | CtIP: A DNA damage response protein at the intersection of DNA metabolism[END_REF], is strongly contracted in PtCtIP, comprising less than 50 amino acids (Fig. 1 and Supplemental Fig. 1). Nonetheless, the miniature PtCtIP is fully functional for HR-mediated repair of meiotic DSBs, emphasizing the interest of this homolog as a model to disentangle the core function of the CtIP-MR machinery from other regulatory functions. C. elegans, and T. thermophila [START_REF] Penkner | A conserved function for a Caenorhabditis elegans Com1/Sae2/CtIP protein homolog in meiotic recombination[END_REF][START_REF] Uanschou | A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene[END_REF][START_REF] Limbo | Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control doublestrand break repair by homologous recombination[END_REF][START_REF] Lukaszewicz | MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena[END_REF]. Furthermore, spore viability of 'CtIP' mutant cells could be partially rescued in a Spo11-deficient background in most species, indicating that it functions downstream of Spo11 in the repair of meiotic DSBs [START_REF] Hartsuiker | Ctp1CtIP and Rad32Mre11 nuclease activity are required for Rec12Spo11 removal, but Rec12Spo11 removal is dispensable for other MRN-dependent meiotic functions[END_REF].

P. tetraurelia contains two paralogous CtIP genes required for completion of meiotic

Due to a recent whole genome duplication event [START_REF] Aury | Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia[END_REF], two closely related CtIP gene copies, PtCtIPa and PtCtIPb, were identified in the model ciliate P. tetraurelia [START_REF] Arnaiz | ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia[END_REF].

Interestingly, we found that PtCtIPa and PtCtIPb gene expression profiles are comparable to that of PtSPO11 and PtMRE11b, displaying highest mRNA transcript levels in early meiosis (Fig. 2A andB). Simultaneous silencing of both PtCtIP genes impaired the recovery of functional new MACs in sexual progeny to a similar extent as Mre11b or Spo11 knockdowns (Fig. 2C), indicative of an essential function of PtCtIP during meiotic recombination.

Specifically, we show that PtCtIP knockdown cells exhibit a defect in the progression of meiosis I with complete absence of new developing MACs at late stages of the sexual cycle (Fig. 3). Strikingly, PtCtIP disruption in a Spo11-deleted strain partially restored the physical development of new, yet non-viable MACs (Fig. 3B and Supplemental Fig. 4A), establishing that PtCtIP is a bona fide ortholog of Sae2/Ctp1/CtIP.

The RHR motif is critical for PtCtIP DNA-binding and DSB resection in human cells

We demonstrated that purified recombinant PtCtIP preferentially recognizes branched (or flap) DNA substrates through a highly conserved RHR motif located at the C-terminus in all CtIP homologs (Fig. 4) [START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF]. Our gel shift assays further suggested that PtCtIP binds to DNA in a multimeric state, giving rise to a high molecular weight complex. These results are in line with structural studies establishing an N-terminal tetrameric oligomerization scaffold in Ctp1 and CtIP [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF][START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF], core residues of which are conserved in the PtCtIP protein sequence.

Remarkably, while being unable to cleave DNA on its own, PtCtIP interacts with recombinant human MR complex, indicative of an evolutionary conserved protein-protein interaction module.

It has been reported that mutations of the RHR motif in Ctp1 resulted in an intermediate level of DNA damage sensitivity in S. pombe strains, suggesting that additional factors can partially compensate for DNA-binding defects [START_REF] Andres | Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA doublestrand-break repair[END_REF]. Here, we provided evidence that mutation

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of the RHR motif in human CtIP abrogates its spatial redistribution in response to DNA damage, supporting the importance of the DNA-binding activity across different species (Fig. 5A). In addition, we observed that human cells expressing a CtIP-AHA DNA-binding mutant display DNA-end resection and HR defects, ultimately resulting in profound hypersensitivity to CPT treatment (Fig. 5B-E). Notably, however, PtCtIP-AHA mutant is proficient for interacting with MR as well as for stimulating MR-dependent DNA cleavage in vitro (Supplemental Fig. 6C-E), suggesting that the DNA-binding function of CtIP proteins is mechanistically separable from MR activation in DSB repair processes. Similarly, it was reported that DNA-binding of Sae2 is not necessary for the stimulation of the MRX endonuclease [START_REF] Cannavo | Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks[END_REF].

In conclusion, our study provides converging evidence that the minimal Paramecium CtIP ortholog, being deprived of most of the regulatory motifs found in other CtIP protein species, has retained the ability to repair Spo11-induced meiotic DSBs. Our combined biochemical and functional analyses further imply that the DNA-binding activity of CtIPrelated proteins is critical for the maintenance of genomic stability, perhaps by promoting the accumulation and correct assembly of CtIP tetramers at DSBs. Clearly, further investigations are required to elucidate how CtIP structurally integrates with the MR complex to promote efficient repair of DSBs. We propose that the trimmed-down PtCtIP might provide an ideal source for high-resolution structural analyses aiming to answer this question.

A C C E P T E D M

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Materials and methods

Bioinformatics analysis of protein sequences

Due to the high sequence divergence among eukaryotic CtIP orthologs, a hierarchical approach was used to build the global alignment. First, six sequence profiles were independently generated from the full sequences of CtIP proteins in vertebrates ( 35sequences), nematodes (10 sequences), plants (16 sequences), arthropods (19 sequences), protozoa (15 sequences) and fungi (63 sequences) using the MAFFT v7.0 algorithm with the E-INS-i iterative refinement method [START_REF] Katoh | MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability[END_REF]. Next, using the HHsearch algorithm [START_REF] Söding | Protein homology detection by HMM-HMM comparison[END_REF], these profiles were hierarchically aligned together resulting in a large multiple sequence alignment of 158 protein sequences. From this alignment, 35 sequences from model organisms were extracted and trimmed so that only the most conserved C-terminal domain was considered for the phylogenetic tree reconstruction. Phylogenetic tree was calculated using the PhyML algorithm [START_REF] Guindon | New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0[END_REF] focusing on the C-terminal domain spanning residues 122-198 of PtCtIP with standard parameters (LG model of amino acids substitution, discrete gamma model with 6 categories and gamma shape factor of 1.729). Representation of the tree was performed using Dendroscope 3 [START_REF] Huson | Dendroscope 3: An Interactive Tool for Rooted Phylogenetic Trees and Networks[END_REF]. Coiled-coil predictions were performed using PCOILS [START_REF] Lupas | Predicting coiled coils from protein sequences[END_REF].

P. tetraurelia strains, cultivation, and gene silencing

Autogamy and conjugation experiments were carried out at 27°C with strain 51new [START_REF] Gratias | Processing of double-strand breaks is involved in the precise excision of paramecium internal eliminated sequences[END_REF]. Cells were grown in a wheat grass powder (WGP, Pines International Inc.) infusion medium bacterized the day before use with Klebsiella pneumoniae and supplemented with 0.8 µg/ml β-sitosterol. The somatic SPO11 knockout was generated as described previously [START_REF] Saudemont | The fitness cost of mis-splicing is the main determinant of alternative splicing patterns[END_REF]. In brief, following conjugation of reactive 51new cells from mating types 7 (mt7) and 8 (mt8), stable mating pairs were transferred to WGP medium inoculated with Escherichia coli HT115 bacteria harbouring plasmid PtSPO11-1 and induced for dsRNA production [START_REF] Baudry | PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF].

For RNAi-mediated gene silencing experiments during autogamy, cells were grown for 20 to 25 vegetative divisions under standard conditions, before transfer to WGP medium inoculated with HT115 bacteria harbouring the appropriate dsRNA-producing plasmids [START_REF] Galvani | RNA interference by feeding in Paramecium[END_REF]. To monitor the progression of autogamy, cells were permeabilized and fixed prior to DAPI staining as described [START_REF] Marmignon | Kumediated coupling of DNA cleavage and repair during programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF]. Nuclear developmental stages were observed using a Zeiss Axioplan 2

Imaging epifluorescence microscope with 63x/1.4 Plan-Apochromate or 40x/1.3 Plan-Neofluar oil objectives. The capability of sexual progeny to develop a functional new MAC was monitored following individual transfer of 30 autogamous cells to standard culture medium [START_REF] Kapusta | Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV-Dependent End Joining[END_REF].

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RNAi experiments during conjugation were performed as described [START_REF] Baudry | PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF]. In brief, reactive 51new mt7 and mt8 cells were prepared in each RNAi medium and mixed to start conjugation. Following 1.75 h incubation at 27°C, freshly induced RNAi medium was added to the mix to synchronize conjugation. Mating pairs were transferred manually to each RNAi medium and incubated at 27°C to complete MAC development and resume vegetative growth.

To monitor sexual processes, conjugating cells were permeabilized, fixed and processed for immunostaining as described [START_REF] Dubois | Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements[END_REF]. To reveal micronuclei and the zygotic nucleus during conjugation, cells were incubated for 1 h with primary anti-γ-tubulin antibodies (1:400, [START_REF] Klotz | Gamma-tubulin and MTOCs in Paramecium[END_REF]), then washed with TBST (10 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.1% Tween) + 3% BSA prior to 15 min incubation with secondary Alexa Fluor 488-conjugated anti-rabbit antibodies

(1:300, ThermoFisher Scientific), followed by DAPI staining. Imaging was performed using a Zeiss Axioplan 2 Imaging epifluorescence microscope with a 63x/1.4 Plan-Apochromate oil objective.

dsRNA-producing plasmids for silencing P. tetraurelia genes

All RNAi plasmids are derivatives of vector L4440 [START_REF] Kamath | Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans[END_REF] and carry a target gene fragment between two convergent T7 promoters. Each insert was chosen in order to minimize the risk of cross-silencing by using the "RNAi off-target" tool of ParameciumDB [START_REF] Arnaiz | ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia[END_REF].

Plasmids p0ND7c [START_REF] Garnier | RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia[END_REF], pICL7a [65] and pL4440-SPO11-1 [START_REF] Baudry | PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF] were used to target Paramecium ND7, ICL7a and SPO11 genes, respectively. RNAi plasmids targeting PtCtIP and PtMRE11 genes were constructed as follows: pL4440-PtCtIPa: a 354-bp fragment from PtCtIPa (nt 266-619) was amplified by PCR from total genomic DNA from P. tetraurelia, using primers PtCtIPa-SpeI-F and PtCtIPa-SpeI-R (Supplemental Table S1) and the Phusion High-Fidelity DNA polymerase (New England Biolabs). Following SpeI-restriction, the PCR fragment was inserted into the unique XbaI site of vector L4440. pL4440-PtCtIPb: same cloning procedure for a 354-bp fragment from PtCtIPb (nt 266-619) amplified using primers PtCtIPb-SpeI-F and PtCtIPb-SpeI-R. pL4440-PtMRE11a1: same cloning procedure for a 492-bp fragment from PtMRE11a (IF2: nt 1422-1913) amplified using primers PtMRE11a1-SpeI-F and PtMRE11a1-SpeI-R. pL4440-PtMRE11b1: same cloning procedure for a 510-bp fragment from PtMRE11b (IF2: nt 1420-1929) amplified using primers PtMRE11b1-SpeI-F and PtMRE11b1-SpeI-R. pL4440-PtMRE11a2: same cloning procedure for a 507-bp fragment from PtMRE11a (IF1: nt 24-530) amplified using primers PtMRE11a2-SpeI-F and PtMRE11a2-SpeI-R. The IF1 sequence from PtMRE11a cross-reacts with PtMRE11b.

pL4440-PtMRE11b2: same cloning procedure for a 508-bp fragment from PtMRE11b (IF1:

A C C E P T E D M A N U S C R I P T
nt 24-531) amplified using primers PtMRE11b2-SpeI-F and PtMRE11b2-SpeI-R. The IF1 sequence from PtMRE11b cross-reacts with PtMRE11a.

Northern blot analysis

Total RNA was extracted from ~400,000 cells for each indicated time point during autogamy of 51new cells grown in standard K. pneumoniae medium and processed for northern blot hybridization using 32 P-labeled probes as described [START_REF] Baudry | PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF]. Unless otherwise stated, PCR fragments of PtSPO11, PtCtIPa, PtCtIPb, PtMRE11a and PtMRE11b RNAitargeting plasmids were used as gene-specific double-stranded probes (Supplemental Table S1). The sequence of the 17S rRNA oligonucleotide probe is shown in Supplemental Table S1. Hybridization signals were collected using a Typhoon TRIO Variable Mode Phosphorimager (GE Healthcare). Quantification was performed using the ImageJ software and mRNA levels were normalized relative to 17S rRNA hybridization signals.

Cloning, expression and purification of recombinant PtCtIPa

The PtCtIPa open reading frame (NCBI database GI accession number 145537105) was synthesized, cloned into pUC57 and verified by sequencing (GeneScript, Piscataway, NJ, USA). PtCtIPa was PCR-amplified using the primers listed in Supplemental Table S1 and cloned into the pFastBac(FB)-MBP-His vector. PtCtIPa-R181A/R183A amino acids substitutions were generated by site-directed mutagenesis using PfuTurbo DNA polymerase (Agilent Technologies) and appropriate primers (Supplemental Table S1). Recombinant PtCtIPa proteins were expressed using the pFB-MBP-PtCtIP-His vectors and the Bac-to-Bac baculovirus expression system (Invitrogen) in insect cells as described previously [66]. In brief, Sf9 cells (1x10 6 cells/ml) cultured in HyClone SFX-Insect cell culture medium (GE Healthcare) were transduced with high-titer viruses and grown for 52 h at 27°C. All subsequent steps were performed at 4°C. Pellets of 200 ml cultures were resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 1 x Halt Protease Inhibitor Cocktail (Thermo Scientific), 1 mM PMSF, 30 µg/ml leupeptin) and incubated stirring for 20 min before adding 0.5 volumes of 50% glycerol. 5 M NaCl was added dropwise to the sample, to reach a final concentration of 300 mM NaCl, and the solution was incubated 30 min before centrifugation at 38,000 g for 30 min. Soluble extracts were bound to pre-equilibrated amylose resin (New England Biolabs, UK) for 1 h and the resin was washed with wash buffer (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 10% Glycerol, 1 mM PMSF, 2 mM β-mercaptoethanol, 10 µg/ml leupeptin). Proteins were eluted with wash buffer containing 10 mM maltose. To

Cloning, expression and purification of recombinant human CtIP-CTD

The CtIP C-terminal domain (CTD) spanning amino acid residues 608-897 was PCRamplified from Flag-CtIP-wt and R837A/R839A plasmids (described in 4.12) using primers listed in Supplemental Table S1 and cloned into pET28-MBP-TEV vector (Addgene: #69929) downstream of the TEV cleavage site. Constructs were expressed in BL21-CodonPlus-RIL E.

coli by growing them for 20 h at 18°C using 0.5 mM IPTG. Bacterial pellets were resuspended in lysis buffer (50 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM zinc acetate) and snap frozen. After thawing, 1 mM PMSF, protease inhibitor cocktail (cOmplete, EDTA-free, Sigma), 2 mM -mercaptoethanol and 0.1 mg/ml lysozyme was added and lysates were stirred at 4°C for 15 min. Subsequently, lysates were sonicated for 5 min and insoluble material was removed by ultracentrifugation at 125,000 g for 1 h. Fusion proteins were purified from clarified lysates through amylose affinity chromatography (5 ml MBPTrap HP,

Size exclusion chromatography (SEC)

Approximately 25 µg of purified PtCtIPa proteins, 50 µg of purified Ctp1 or 50 µg of purified MBP (Prospec) were loaded at 0.5 ml/min onto a Superose 6 10/300 GL size exclusion chromatography column (GE Healthcare) in 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM PMSF and 5 mM β-mercaptoethanol using an ÄKTA™ Pure chromatography system (GE Healthcare).

DNA substrates

The sequences of 50mer oligonucleotides used in DNA binding and nuclease assays are listed in Supplemental Table S1. Where indicated, the 5' end was labeled with T4 polynucleotide kinase (PNK; New England Biolabs) in the presence of [γ-32 P]-ATP, while 3' end-labeling was performed with terminal deoxynucleotidyl transferase (TdT; New England Biolabs) in the presence of [α-32 P]-cordycepin-5'-triphosphate. Unincorporated nucleotides were removed using MicroSpin G25 columns (GE Healthcare). The double-stranded oligo substrates were annealed by heating the oligonucleotides at 95°C and slow gradual cooling to room temperature in either 1x TdT or PNK buffer.

DNA binding and competition assays

All steps were performed at 4°C under native conditions. Recombinant proteins were incubated with 10 nM of the indicated radiolabeled DNA substrates for 20 min at 20°C in 20 µl binding buffer (20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5 mM DTT, 10 µg/ml BSA, 4% glycerol). Protein-DNA complexes were separated on a 4-20% TBE precast gel (Novex) for 120 min at 100 V, respectively, dried and exposed to a phosphor screen before imaging using a Typhon FLA 9500 scanner (GE Healthcare). Data were quantified with ImageQuant TL software (GE Healthcare). In competition experiments, PtCtIPa proteins were incubated with radiolabeled DNA substrates for 5 min on ice prior to the addition of increasing amounts of cold, blunt-ended competitor 61mer dsDNA (Supplemental Table S1). DNA binding assays with recombinant human CtIP-CTD proteins were performed as previously described [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF]. In brief, CtIP-CTD-wt and -AHA mutant were incubated with The pcDNA5/FRT/TO-GFP-CtIP-wild type (wt) and 3x Flag-CtIP-wt expression vectors were described previously [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF][START_REF] Ferretti | Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection[END_REF]. CtIP-R837A/R839A amino acids substitutions were generated by site-directed mutagenesis using PfuTurbo DNA polymerase and appropriate primers (Supplemental Table S1). All CtIP constructs are siRNA-resistant [START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF]. Antibodies for immunoblotting were: anti-FLAG M2 (Sigma F3165, 1:1000), anti-GFP (Santa Cruz B-2, 1:1000), anti-Mre11 (GeneTex 12D7, 1:1000), anti-Rad50 (GeneTex 13B3, 1:1000), anti-TFIIH p89 (Santa Cruz S-19, 1:1000), anti-RPA2 (Calbiochem NA19L, 1:1000), antiphospho RPA2 (S4/S8) (Bethyl A300-245A, 1:5000), anti-phospho ATM (S1981) (Abcam ab81292, 1:5000). Control siRNA (luciferase 5′-CGUACGCGGAAUACUUCGA-3′) and

CtIP siRNA (5′-GCUAAAACAGGAACGAAUC-3′) [START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF] were purchased from Microsynth. induction of protein expression with 1 µg/ml doxycycline (Dox, Sigma-Aldrich). The CtIP-R837A/R839A mutation in stable U2OS cells was verified by genomic sequencing. U2OS-TLR cells [START_REF] Davies | CtIP tetramer assembly is required for DNA-end resection and repair[END_REF] stably transfected with Flag-tagged versions of CtIP were grown in the presence of 0.5 μg/ml puromycin and 0.5 mg/ml geneticin. Plasmid transfections were performed using the Fugene 6 transfection reagent (Promega) according to the manufacturer's instructions. siRNA transfections were performed using Lipofectamine RNAiMax (Life Technologies) according to the manufacturer's instructions. Camptothecin (CPT) was purchased from Sigma. Laser micro-irradiation was performed as described previously [START_REF] Ferretti | Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection[END_REF].

In brief, cells were grown in medium supplemented with 10 µM BrdU for 24 h. Cells were microirradiated using a MMI CELLCUT system containing a UVA laser of 355 nM (Molecular Machines and Industries). The laser intensity was set to 50% energy output and each cell was exposed to the laser beam for 300 ms.

Immunofluorescence microscopy

Cells grown on coverslips were fixed directly in formaldehyde (4%, w/v in PBS) for 15 min and permeabilized for 5 min in 0.5% Triton X-100 in PBS. Cells were incubated for 1 h with primary anti-H2AX-pS139 antibody (Cell Signalling 20E3, 1:500), followed by 1 h incubation with secondary Alexa Fluor 594-conjugated anti-rabbit antibody (Life Technologies, 1:1000). Coverslips were mounted with Vectrashield ® (Vector Laboratories) containing DAPI and imaged using a Leica DM6B fluorescence microscope at 63X magnification.

DNA-end resection assay

Flow cytometry-based resection assay was performed as described previously [START_REF] Ferretti | Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection[END_REF].

Briefly, U2OS cells were transfected with CtIP siRNA. Where indicated, 24 h posttransfection, doxycycline was added to the cells to induce the expression of GFP-CtIP. 48 h post-transfection, cells were either mock-treated or treated for 1 h with 1 μM CPT. Cells were harvested, pre-extracted with 0.3% Triton-X100 in PBS for 15 min on ice and fixed with 4% formaldehyde (w/v) for 10 min at room temperature. Cells were incubated for 1 h with anti-RPA2 antibody (Calbiochem NA19L, 1:100) or anti-H2AX-pS139 antibody (Cell Signalling 20E3, 1:200), followed by 30 min incubation with Alexa Fluor 647-or 488-conjugated secondary antibody (Life Technologies, 1:250) and counterstained with DAPI/RNase.

Samples were analysed by flow cytometry on a CyAn ADP 9 (Dako). Data analysis was performed using FlowJo X software (Tree Star).

CellTiter-Blue ® Cell viability assay

Cell viability was measured as previously described [START_REF] Ferretti | Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection[END_REF]. Briefly, U2OS clones stably expressing doxycycline-inducible siRNA-resistant forms of GFP-CtIP were transfected with indicated siRNAs. 24 h post-transfection, cells were seeded in triplicates at a density of 500 cells/well in a 96-well plate in medium supplemented with 1 µg/ml doxycycline. 24 h later, cells were continuously treated with indicated doses of CPT and grown for 4 days at 37°C.

To measure viability, CellTiter-Blue ® reagent (Promega) was added on the last day and incubated for 4 h at 37°C before fluorescence was measured at 560/590 nm using a microplate reader (Molecular Devices).

  /CtIP protein family members that have been identified so far.

Fig. 1 :

 1 Fig. 1: Comparative protein sequence analysis of PtCtIP. (A) Upper panel, predicted protein scheme of Paramecium tetraurelia CtIP (PtCtIP) with putative coiled-coil (CC) and C-terminal regions indicated in coloured boxes. Lower panel, coiled-coil-forming probabilities predicted by the PCOILS program (window length of 21 residues) for the indicated CtIP proteins. (B) Phylogenetic tree obtained from the sequence analysis of the CtIP C-terminal region from 35 species using PhyML algorithm. (C) Sequence alignment of the Cterminal region of PtCtIPa and PtCtIPb with 12 CtIP proteins from different species. Gene accession numbers (gi) and species names are color-coded according to the phylogenetic tree shown in (B). Insertions and extensions were trimmed and marked by dashed lines with the number of residues indicated below. Evolutionarily conserved CxxC, RHR and S/TP motifs are indicated in black boxes.

  autogamy of cells silenced for the expression of both PtCtIPa and b genes, the development of new MACs in the sexual progeny was monitored after DAPI staining. Strikingly, in all

Fig. 2 :

 2 Fig. 2: PtCtIP genes are required for recovery of viable sexual progeny. (A) Steady-state levels for PtCtIP, PtSPO11 and PtMRE11 mRNA at different developmental stages. Curves show the average normalized values calculated for each autogamy stage (n  2). RNA-Seq data extracted from [42]. Veg: vegetative cells; Mei: starved cells with meiotic stages; Frag: fragmented old MAC but no visible developing new MACs; Early: early stages of MAC development; Inter: intermediate stages of MAC development; Late: late stages of MAC development. (B) Quantification of PtCtIPa and PtCtIPb mRNA levels during autogamy. P. tetraurelia 51 cells were subjected to control RNAi (ND7) or RNAi against both PtCtIPa and PtCtIPb (PtCtIPa+b). Autogamy stages were defined as in (A) following DAPI staining of nuclei. Northern blots of total RNAs extracted at indicated time-points for each RNAi condition were hybridized with PtCtIPa-or PtCtIPb-specific labeled probes A2 (upper panel) or B2 (lower panel), respectively (Supplemental Fig. 2C and Supplemental TableS1). Viable post-autogamous progeny yields in this experiment: 97%, control RNAi; 23%, PtCtIPa+b RNAi. 17S rRNA signal was used for normalization. Y-axes are in arbitrary units, with CTRL mRNA signals normalized to 1 at the Mei stage for each probe. (C) Combined depletion of PtCtIPa and PtCtIPb impairs new MAC development in Paramecium sexual progeny. Histograms show the fraction of sexual progeny harboring functional new MACs after silencing of the respective genes by RNAi. Data are presented as the mean ± SD with n between 2 and 15 for each condition. Statistical differences were determined by paired t-test. ns, nonsignificant; ** (P < 0.005); **** (P < 0.0001). CNTL: RNAi-mediated targeting of either ND7 or ICL7 nonessential genes. For simultaneous silencing of both PtMRE11 genes, different constructs were used (crossreacting a2 and b2 constructs, either alone or together; gene-specific a1 and b1 constructs, together; see Supplemental TableS1).

Fig. 3 :

 3 Fig. 3: PtCtIP silencing impairs new MAC development due to abortive meiosis. (A) PtCtIPRNAi during conjugation triggers a meiotic defect and impairs zygotic nucleus formation. Reactive cells prepared in control (ND7) or PtCtIPa+b RNAi medium were crossed at T0 and mating pairs were permeabilized and fixed 4.5, 6 and 7.5 hours following the start of conjugation. Fixed cells were pooled before proceeding to immunofluorescence staining using anti-γ-tubulin antibodies (green) and DAPI staining of nuclei (separate channels of the same images are shown in Supplemental Fig.3). The progression of conjugation was monitored according to the state of the old MAC. Arrowheads (yellow in one partner, white in the other) point to meiotic micronuclei, or to zygotic nuclei and their mitotic products. 22 and 30 mating pairs were analyzed for the control (ND7) and for PtCtIP RNAi, respectively. Numbers in orange refer to pairs in which loss of micronuclei ('Mic loss') was detected in one or both partners. In the experiment shown here, 37% of mating pairs yielded viable progeny in the PtCtIP RNAi (100% in the control RNAi). (B) MAC development is inhibited following PtCtIPa+b RNAi in wild-type cells and restored at the cytological level in a SPO11 knockout Paramecium strain submitted to PtCtIPa+b RNAi. Cultures of isogenic wild-type and ΔPtSPO11 cells were allowed to undergo autogamy in control (ND7) or PtCtIP RNAi-inducing medium. The histograms show the progression of autogamy in the four cultures, as monitored by DAPI staining of cells at indicated time-points (hours), with the A C C E P T E D M A N U S C R I P T

  substrate, confirming the C-terminal 'RHR' motif as a critical determinant of DNA-binding by

Fig. 4 :

 4 Fig. 4: PtCtIP and human CtIP require a highly conserved 'RHR' motif for DNA-binding. (A) PtCtIP binds double-stranded (ds) DNA. Electrophoretic mobility shift assay (EMSA) was carried out with 10 nM of various 5'-radiolabeled DNA substrates and 600 nM of PtCtIPa. Percentages of DNA binding were calculated based on the disappearance of the substrate band. (B) PtCtIP has a slight preference for forked DNA. Upper panel, EMSA was carried out with 10 nM of the indicated 5'-radiolabeled DNA substrates and increasing concentrations of PtCtIPa (100 nM, 200 nM, 400 nM, 700 nM and 1 µM). Ctp1 (380 nM) was used as a positive control. Lower panel, Percentages of DNA binding were calculated based on the disappearance of the substrate band. Data are represented as the mean ± SD (n = 3). (C) Alignment of the amino acid sequences adjoining the conserved RHR motif in CtIP homologs from P. tetraurelia (Pt), S. pombe (Sp) and H. sapiens (Hs). The R181A/R183A and R837A/R839A (AHA) mutations in PtCtIP and HsCtIP are indicated above and below the alignment, respectively. (D) Mutation of the RHR motif in PtCtIP abolishes DNA binding. EMSA was carried out with 10 nM of 5'-radiolabeled forked DNA substrates and increasing concentrations of PtCtIPa wild-type (wt) or AHA mutant (200 nM, 400 nM, 600 nM, 800 nM and 1 µM). Ctp1 (380 nM) was used as positive control. Percentages of DNA binding were calculated based on the disappearance of the substrate band. (E) Mutation of the RHR motif in human CtIP impairs binding to dsDNA. Upper panel, EMSA was carried out with 1 µM fluorescein (F)-labeled, blunt-ended dsDNA in presence of increasing concentrations of recombinant human CtIP C-terminal domain (CTD; 1 µM, 5 µM, 10 µM, 20 µM, 50 µM)). Lower panel, Percentages of shifted DNA in each lane were calculated by quantifying the fluorescence intensity of shifted relative to total DNA. Data are represented as the mean ± SD (n = 3).

Fig. 5 :

 5 Fig. 5: Mutation of the RHR motif impairs CtIP accumulation at DSBs, DNA-end resection and HR. (A) U2OS clones harbouring inducible wt and AHA mutant GFP-CtIP were transfected with siCtIP for 48 h. 24 h later, cells were grown on coverslips in the presence of doxycycline (Dox) and BrdU for 24 h prior to laser micro-irradiation. 20 min post-irradiation, cells were fixed, immunostained for γH2AX and counterstained with DAPI for DNA and analysed by fluorescence microscopy. Upper panel, representative images. Scale bar, 10 µM. Lower panel, scatter plot of relative fluorescent intensities of GFP-CtIP laser stripes normalized to γH2AX stripe intensity (see Supplemental Fig. 6C). Data are presented as the mean (indicated by red bars) ± SD. Statistical difference was determined by Mann-Whitney test; **** (P < 0.0001). For each condition, more than 20 cells from two independent experiments were quantified. (B) The same cells as described in (A) transfected with control (CNTL) or CtIP siRNA were cultivated for 24 hr in absence (-) or presence (+) of Dox. 48 h post siRNA-transfection, cells were mock-treated (lane 1) or treated with CPT (1 µM) for 1 h and whole cell lysates were analysed by immunoblotting using the indicated antibodies. Asterisks indicate hyperphosphorylated forms A C C E P T E D M A N U S C R I P T

  An essential role for CtIP-related proteins in DSB repair was first described in S. cerevisiae, where Sae2 acts in concert with MRX to remove Spo11-oligonucleotide complexes from DSB ends during meiosis[12,13]. Consequently, Mre11 nuclease-deficient and sae2∆ mutant yeast strains fail to sporulate. Similar meiotic recombination defects were later observed for CtIP mutants in S. pombe, A. thaliana,

  cleave the MBP tag, samples were incubated for 3 h with recombinant prescission protease (PP) expressed in E. coli and purified using standard procedures[START_REF] Anand | Phosphorylated CtIP Functions as a Cofactor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection[END_REF]. Next, imidazole was added to a final concentration of 20 mM before adding 0.5 ml Ni-NTA-agarose (Qiagen). The resin was extensively washed with NTA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5 mM PMSF, 2 mM β-mercaptoethanol, 10 mM imidazole) and proteins eluted with NTA buffer supplemented with 400 mM imidazole. Peak fractions containing PtCtIPa-His were incubated with amylose resin to remove the remaining uncleaved MBPtagged PtCtIPa, pooled, and dialysed against dialysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% glycerol, 0.5 mM PMSF and 5 mM β-mercaptoethanol). Small aliquots of the purified PtCtIPa proteins were snap frozen and stored at -80°C. Recombinant human CtIP and Mre11-Rad50 (MR) complex were expressed and purified as described previously[67].

  Healthcare) and elution with 20 mM Tris pH 8.0, 2 mM -mercaptoethanol, 300 mM NaCl, 2 M methyl -D-glucopyranoside (Sigma). Prior to cleavage with TEV protease, buffer exchange into 20 mM Tris pH 8.0, 300 mM NaCl, 5 mM -mercaptoethanol was performed using a HiPrep 26/10 Desalting column (GE Healthcare). Proteins were concentrated to 2 mg/ml using Amicon Ultra 10'000 MWCO centrifugal filter units (Millipore) and MBP-tagged TEV protease (Gene and Cell Technologies) was added in a ratio of 1:5. Cleavage was performed overnight at 20°C and unwanted protein species were captured by amylose affinity chromatography. Further purification of amylose chromatography flow-through was achieved through size-exclusion chromatography (HiLoad Superdex 75, GE Healthcare) in 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM mercaptoethanol.

4. 13 .

 13 Cell lines, cell culture, transfections and treatments U2OS cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin. The Flp-In T-REx system (Invitrogen Life Technologies) was used to generate U2OS cells stably expressing siRNA-resistant GFP-tagged CtIP-wt or CtIP-R837A/R839A under the control of a doxycycline-inducible promoter. In brief, pcDNA5/FRT/TO-GFP-CtIP and the Flp recombinase expression plasmid, pOG44, were mixed in a 1:9 ratio and transfected into U2OS Flp-In T-REx cells. 24 h later, cells were plated at different dilutions and 48 h post-transfection the medium was supplemented with 250 mg/ml hygromycin B and 12.5 mg/ml blasticidin S. The medium was replaced every 2-3 days and cells were selected for approximately 14 days. Resistant colonies were picked and single-cell clones analysed for GFP expression by immunoblotting and immunofluorescence microscopy after 24 h

A

  AH, Kleckner N. A general method for identifying recessive diploid-specific mutations in Saccharomyces cerevisiae, its application to the isolation of mutants blocked at intermediate stages of meiotic prophase and characterization of a new gene SAE2. Genetics. 1997;146: 797-816. 13. Prinz S, Amon A, Klein F. Isolation of COM1, a new gene required to complete Functional diversification of centrins and cell morphological complexity. J Cell Sci. 2008;121: 65-74. doi:10.1242/jcs.019414 66. Cejka P, Kowalczykowski SC. The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions. J Biol Chem. 2010;285: 8290-8301. doi:10.1074/jbc.M109.083196 67. Zheng L, Kanagaraj R, Mihaljevic B, Schwendener S, Sartori AA, Gerrits B, et al. MRE11 complex links RECQ5 helicase to sites of DNA damage. Nucleic Acids Res. 2009;37: 2645-2657. doi:10.1093/nar/gkp147

). RNA-Seq data extracted from [42]. Veg: vegetative cells; Mei: starved cells with meiotic stages; Frag: fragmented old MAC but no visible developing new MACs; Early: early

  stages of MAC development; Inter: intermediate stages of MAC development; Late: late stages of MAC development. (B) Quantification of PtCtIPa and PtCtIPb mRNA levels during autogamy. P. tetraurelia 51 cells were subjected to control RNAi (ND7) or RNAi against both PtCtIPa and PtCtIPb (PtCtIPa+b). Autogamy stages were defined as in (A) following DAPI staining of nuclei. Northern blots of total RNAs extracted at indicated time-points for each RNAi condition were hybridized with PtCtIPa-or PtCtIPb-specific labeled probes A2 (upper panel) or B2 (lower panel), respectively (Supplemental Fig. 2C and Supplemental Table S1). Viable post-autogamous progeny yields in this experiment: 97%, control RNAi; 23%, PtCtIPa+b RNAi. 17S rRNA signal was used for normalization. Y-axes are in arbitrary units, with CTRL mRNA signals normalized to 1 at the Mei stage for each probe. (C) Combined depletion of PtCtIPa and PtCtIPb impairs new MAC development in Paramecium sexual progeny. Histograms show the fraction of sexual progeny harboring functional new MACs after silencing of the respective genes by RNAi. Data are presented as the mean ± SD with n between 2 and 15 for each condition. Statistical differences were determined by paired t-test. ns, non-

significant; ** (P < 0.005); **** (P < 0.0001). CNTL: RNAi-mediated targeting of either ND7 or ICL7 nonessential genes. For simultaneous silencing of both PtMRE11 genes, different constructs were used (crossreacting a2 and b2 constructs, either alone or together; gene-specific a1 and b1 constructs, together; see Supplemental Table

S1

).

  in 0.5 x TBE buffer at 25 V for 3 h at 4°C. DNA was visualized using Typhoon FLA 9500 FluorImager. Data were quantified with ImageJ software.

	4.10. DNA cleavage assays
	Recombinant proteins were incubated with 10 nM of 5' or 3' radiolabeled DNA
	substrates for 60 min at 37°C in 10 µl reaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM
	NaCl, 0.5 mM DTT, 10 µg/ml BSA, 4% glycerol) supplemented with either 5 mM MgCl2 or
	1 mM MnCl2. 10 nM of purified recombinant human DNA2 was used as a control. Nuclease reactions were stopped by adding an equal volume of 80% formamide loading dye. Samples were boiled for 5 min and products were separated on a 20% polyacrylamide gel containing 7 M urea. Gels were fixed in 1x TBE buffer containing 10% acetic acid, 50% EtOH and 10% glycerol for 20 min, dried and exposed to a phosphor screen before imaging on a Typhoon FLA 9500 scanner (GE Healthcare). Nuclease assays using PhiX174 single-stranded circular S C R I P T (ssc) DNA as a substrate was performed as described previously [14]. In brief, 85 ng of U PhiX174 virion DNA was incubated with recombinant proteins for 2 h at 37°C in 10 µl
	N reaction buffer (30 mM potassium/MOPS, pH 7.2, 1 mM DTT, 1 mM ATP, 25 mM KCl, 25
	ng/ul BSA) supplemented with 5 mM of either MgCl2 or MnCl2. Reactions were stopped by A adding 0.5 μl Proteinase K (20.6 mg/ml, Roche) and 4 μl stop solution (2% SDS, 50 mM M EDTA) for 20 min at room temperature. Samples were run on a 0.8% TAE agarose gel for 1
	h at 80 V, stained with SYBR Gold Nucleic Acid Gel Stain (Molecular Probes) for 20 min and the gel was imaged using a Typhoon FLA 9500 scanner. Data were quantified with ImageQuant TL software. 4.11. MBP pull-down assay Soluble protein extracts of Sf9 cells expressing MBP alone or MBP-PtCtIPa-His were incubated with amylose resin for 1 h at 4°C. Resin was washed three times with NTEN buffer (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.5% NP-40) containing 300 mM NaCl and once C C E P T E D with TEN100 buffer (20 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 100 mM NaCl). MBP
	A proteins coupled to amylose beads were mixed with 0.5 µg of recombinant human Mre11-
	Rad50 (MR) complex for 2 h at 4°C. Beads were washed with NTEN buffer containing 500
	mM NaCl and proteins were eluted in TEN100 buffer containing 20 mM maltose. Samples
	were analysed by SDS-PAGE followed by immunoblotting.

1 M fluorescein-labeled 50mer dsDNA for 1 h at 21°C and loaded onto 1.2 % agarose gel (in 0.5x TBE buffer). Gel was run 4.12. Plasmids, antibodies and siRNA

Acknowledgements

We would like to thank Cindy Mathon and Pascaline Tirand for excellent technical assistance and Loïc Escoriza, Nathalie Mathy and Coralie Zangarelli for their help in the handling of Paramecium cultures. Anti-γ-tubulin antibodies were kindly provided by Janine Beisson and Anne-Marie Tassin (I2BC, Gif-sur-Yvette, France). We thank Pavel Janscak (University of Zurich, Switzerland) for providing purified recombinant human CtIP and Mre11-Rad50 complex and for experimental advice. We thank Petr Cejka (Institute for Research in Biomedicine, Bellinzona, Switzerland) for providing pFastBac-MBP-His vector,

Funding

This work was supported by the Swiss National Science Foundation [grant numbers 31003A_135507, 31003A_156023, and 31003A_176161, all to A.A.S.]; the Swiss Cancer League [grant number KFS-3845-02-2016-R to A.A.S.]; the Centre National de la Recherche Scientifique (CNRS) [intramural funding to M.B.], the ARC Foundation for Cancer Research [grant number SFI20121205487 to M.B.] and the French National Agency for Research (ANR) [grant number ANR-14-CE10-0005-01 to M.B., grant number ANR-15-CE110008-01 to R.G.]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest statement

The authors declare that there is no conflict of interest.