Aurore Sanchez

Céline Adam

Felix Rauh

Yann Duroc

Lepakshi Ranjha

Bérangère Lombard

Xiaojing Mu

Mélody Wintrebert

Damarys Loew

Alba Guarné

Stefano Gnan

Chun-Long Chen

Scott Keeney

Petr Cejka

Raphaël Guérois

Franz Klein

Jean-Baptiste Charbonnier

Valérie Borde
email: valerie.borde@curie.fr

Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation

Crossovers generated during the repair of programmed meiotic double-strand breaks must be tightly regulated to promote accurate homolog segregation without deleterious outcomes such as aneuploidy. The Mlh1-Mlh3 (MutLγ) endonuclease complex is critical for crossover resolution, which involves mechanistically unclear interplay between MutLγ and Exo1 and Polo kinase Cdc5.

Using budding yeast to gain temporal and genetic traction on crossover regulation, we find that MutLγ constitutively interacts with Exo1. Upon commitment to crossover repair, MutLγ-Exo1 associate with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity. We propose that Exo1 serves as a central coordinator in this molecular interplay, providing a defined order of interaction that prevents deleterious, premature activation of crossovers. MutLγ associates at a lower frequency near centromeres, indicating that spatial regulation across chromosomal regions reduces risky crossover events. Our data elucidate the temporal and spatial control surrounding a constitutive, potentially harmful, nuclease. We also reveal a critical, non-catalytic role for Exo1, through non-canonical interaction with Polo kinase. These mechanisms regulating meiotic crossovers may be conserved across species.

Significance

Meiotic crossovers are essential for the production of gametes with balanced chromosome content. MutLγ (Mlh1-Mlh3) endonuclease is a mismatch repair heterodimer that functions also during meiosis to generate crossovers. Its activity requires Exo1 as well as the MutSγ heterodimer (Msh4-Msh5). Crossovers also require the polo kinase Cdc5, in a way that is poorly understood. We show that Exo1 directly interacts with Cdc5, and that this promotes the activation of MutLγ for crossovers. Since Cdc5 also controls other key meiotic processes, including kinetochores mono-orientation and disassembly of the synaptonemal complex, this direct interaction with the crossover proteins may be a way to coordinate meiotic chromosome segregation events, to avoid untimely activation of the MutLγ endonuclease once recruited to future crossover sites.

Introduction

Meiotic recombination provides crossovers that allow homologous chromosomes to be transiently physically attached together and then properly segregated [START_REF] Hunter | Meiotic Recombination: The Essence of Heredity[END_REF]. A lack or altered distribution of meiotic crossovers is a major source of aneuploidies, leading to sterility or disorders such as Down syndrome, providing strong impetus for understanding how crossovers are controlled during meiosis. Meiotic recombination is triggered by the formation of programmed DNA double-strand breaks (DSBs), catalyzed by Spo11 together with several conserved protein partners [START_REF] Lam | Mechanism and Regulation of Meiotic Recombination Initiation[END_REF]. Following Spo11 removal, 5' DSB ends are resected and the 3' ends invade a homologous template, leading to formation of D-loop intermediates.

After DNA synthesis, many of these intermediates are dismantled by helicases and repaired without a crossover [START_REF] Muyt | BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism[END_REF]. However, a subset are stabilized, and upon capture of the second DSB end, mature into a double Holliday junction (dHJ), which is almost exclusively resolved as a crossover [START_REF] Allers | Differential timing and control of noncrossover and crossover recombination during meiosis[END_REF][START_REF] Hunter | The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination[END_REF]. Essential to this crossover pathway are a group of proteins, called ZMMs, which collectively stabilize and protect recombination intermediates from helicases [START_REF] Muyt | BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism[END_REF][START_REF] Jessop | Meiotic chromosome synapsis-promoting proteins antagonize the anti-crossover activity of Sgs1[END_REF][START_REF] Oh | BLM ortholog, Sgs1, prevents aberrant crossing-over by suppressing formation of multichromatid joint molecules[END_REF][START_REF] Pyatnitskaya | Crossing and zipping: molecular duties of the ZMM proteins in meiosis[END_REF]. The mismatch repair MutLγ (Mlh1-Mlh3) heterodimer is proposed to subsequently act on these ZMM-stabilized dHJs to resolve them into crossovers [START_REF] Zakharyevich | Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase[END_REF]. Accordingly, MutLγ foci reflect the number and distribution of crossovers on both mammalian and plant meiotic chromosomes (reviewed in [START_REF] Gray | Control of Meiotic Crossovers: From Double-Strand Break Formation to Designation[END_REF]).

In a distinct process, eukaryotic mismatch repair, a MutS-related complex (MutS or MutS) recognizes a mismatch, and recruits a MutL-related complex, to initiate repair together with PCNA and Exo1 (reviewed in [START_REF] Manhart | Roles for mismatch repair family proteins in promoting meiotic crossing over[END_REF]). MutL represents the major mismatch repair activity, which involves its endonuclease activity [START_REF] Gueneau | Structure of the MutLalpha C-terminal domain reveals how Mlh1 contributes to Pms1 endonuclease site[END_REF][START_REF] Kadyrov | Saccharomyces cerevisiae MutLalpha is a mismatch repair endonuclease[END_REF].

The related MutLγ is instead essential to meiotic crossover, a process that remains relatively ill-defined, though MutLγ endonuclease activity is required for crossover [START_REF] Zakharyevich | Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase[END_REF][START_REF] Nishant | A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae[END_REF][START_REF] Ranjha | The Saccharomyces cerevisiae Mlh1-Mlh3 heterodimer is an endonuclease that preferentially binds to Holliday junctions[END_REF][START_REF] Rogacheva | Mlh1-Mlh3, a meiotic crossover and DNA mismatch repair factor, is a Msh2-Msh3-stimulated endonuclease[END_REF]. Despite its importance, and cross-species relevance, the mechanism of dHJ resolution and crossover formation by MutLγ remains unknown. MutLγ alone does not resolve HJs in vitro [START_REF] Ranjha | The Saccharomyces cerevisiae Mlh1-Mlh3 heterodimer is an endonuclease that preferentially binds to Holliday junctions[END_REF][START_REF] Rogacheva | Mlh1-Mlh3, a meiotic crossover and DNA mismatch repair factor, is a Msh2-Msh3-stimulated endonuclease[END_REF][START_REF] Manhart | The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans[END_REF], and may need additional partners, meiosis-specific post-translational modifications and/or a specific DNA substrate to promote specific nuclease activity and hence crossover formation.

Based on recent in vitro experiments and genome-wide analysis of meiotic recombination, it seems that rather than acting as a canonical resolvase, MutLγ may nick the DNA, which could result in dHJ resolution and crossover formation if two closely spaced nicks on opposite strands are made [START_REF] Manhart | The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans[END_REF][START_REF] Marsolier-Kergoat | Mechanistic View and Genetic Control of DNA Recombination during Meiosis[END_REF]. In vitro analyses with recombinant proteins also suggest that MutLγ may need to extensively polymerize along DNA in order to activate its DNA cleavage activity [START_REF] Manhart | The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans[END_REF].

MutLγ partners with the ZMM MutSγ heterodimer (Msh4-Msh5), proposed to first stabilize recombination intermediates and then to work together with MutLγ for crossover resolution (reviewed in [START_REF] Pyatnitskaya | Crossing and zipping: molecular duties of the ZMM proteins in meiosis[END_REF][START_REF] Manhart | Roles for mismatch repair family proteins in promoting meiotic crossing over[END_REF]). In addition, the exonuclease Exo1 interacts in vitro with Mlh1. Previous work showed that Exo1's ability to interact with Mlh1 is essential for MutLγ function in crossover formation [START_REF] Zakharyevich | Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions[END_REF]. This leaves the mechanism and role of Exo1 in meiotic crossovers unclear, although broad genetic analysis suggests a non-catalytic role given that Exo1 null yeast show reduced crossovers and reduced viable gametes, but catalytic mutants of Exo1 do not [START_REF] Zakharyevich | Delineation of joint molecule resolution pathways in meiosis identifies a crossover-specific resolvase[END_REF][START_REF] Tsubouchi | Exo1 roles for repair of DNA double-strand breaks and meiotic crossing over in Saccharomyces cerevisiae[END_REF]. Likewise, Exo1-null mice are sterile, but not Exo1 catalytic mutants, highlighting the conservation of this function [START_REF] Schaetzlein | Mammalian Exo1 encodes both structural and catalytic functions that play distinct roles in essential biological processes[END_REF][START_REF] Wei | Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility[END_REF]. How Exo1 is required for activating MutLγ is not known. Finally, the polo-like kinase Cdc5 (homolog of human PLK1) is required for meiotic crossovers [START_REF] Sourirajan | Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis[END_REF]. Cdc5 expression is induced by the Ndt80 transcription factor at the pachytene stage of meiotic prophase, once homolog synapsis and recombination intermediates formation are achieved [START_REF] Chu | The transcriptional program of sporulation in budding yeast[END_REF][START_REF] Chu | Gametogenesis in yeast is regulated by a transcriptional cascade dependent on Ndt80[END_REF][START_REF] Tung | Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae[END_REF][START_REF] Xu | NDT80, a meiosisspecific gene required for exit from pachytene in Saccharomyces cerevisiae[END_REF]. Cdc5 promotes multiple steps during exit from pachytene, including dHJ resolution, disassembly of the synaptonemal complex, and sister kinetochore mono-orientation [START_REF] Clyne | Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I[END_REF][START_REF] Lee | Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation[END_REF][START_REF] Lee | Polo kinase--meiotic cell cycle coordinator[END_REF]. Among other targets, Cdc5 activates the Mus81-Mms4 structure-specific nuclease, by phosphorylating Mms4 [START_REF] Matos | Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis[END_REF], but this produces only a minority of crossovers. It is not known how Cdc5 promotes the remaining crossovers thought to come from the major, MutLγ-dependent, pathway.

In order to define how these factors come together to control crossovers, we undertook a comprehensive analysis of MutLγ activation and distribution genome-wide in budding yeast. We found that MutLγ and Exo1 form a constitutive complex that associates with recombination sites stabilized by ZMM proteins.

Exo1 then interacts with the Cdc5 kinase through a direct, specific contact that is necessary for crossover formation by MutLγ. Finally, we find that MutLγ-dependent crossover formation is regulated at multiple levels to avoid regions around centromeres where deleterious outcomes could result.

Results

Mlh3 foci on yeast meiotic chromosomes are distinct from ZMM foci

To follow how MutL is initially recruited, we examined its relationship to ZMM proteins, Zip3 and Msh4. A recent crystal structure of the C-terminal domain (Cter) of Mlh1-3 shows that the last residues of Mlh1 are part of the Mlh3 endonuclease active site, rendering it impossible to C-terminally tag Mlh1 (Fig. 1A) [START_REF] Duroc | Concerted action of the MutLbeta heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion[END_REF]. To preserve Mlh1-3 functionality in vivo, we introduced an internal tag at an unstructured loop in Mlh3 that is predicted not to affect Mlh1-3 function and catalytic activity (Fig. 1A). Tags inserted at this site had no major effect on mismatch repair and no effect on spore viability or meiotic crossover frequency, and are therefore suitable for further in vivo studies (Fig. S1).

As in plants and mammals, Mlh3 formed foci on yeast meiotic chromosomes (Fig. 1B-E

Mlh3 associates with meiotic DSB hotspots at a late step of recombination

We next examined Mlh3 association with specific loci by chromatin immunoprecipitation. Mlh3 associated with the three tested DSB hotspots during meiosis, reaching a maximum at the expected time of crossover formation (4-5 h) (Figs. 2A and S2A) [START_REF] Hunter | The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination[END_REF], with kinetics very similar to the ZMM proteins [START_REF] Muyt | A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation[END_REF][START_REF] Serrentino | Differential association of the conserved SUMO ligase Zip3 with meiotic double-strand break sites reveals regional variations in the outcome of meiotic recombination[END_REF].

Mlh3 also weakly associated with the chromosome axis, where DSB sites transiently relocate during recombination [START_REF] Panizza | Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination[END_REF][START_REF] Zickler | Meiotic chromosomes: integrating structure and function[END_REF] (Fig. 2A). Mlh3 association with DSB hotspots required DSB formation, since it was not observed in a spo11∆ mutant (Figs. 2A andS2B), but was independent of its nuclease activity (mlh3-D523N mutant, Figs. 2A andS2C). Mlh3 binding also required the ZMM protein, Mer3 (Figs. 2A andS2D). In msh4∆ mutants, Mlh3 recruitment was strongly reduced (Figs. 2A andS2E), consistent with the reduced number of Mlh3 foci in msh4∆ (Figs. 1G and1H). By contrast, Mlh3 bound at hotspots at nearly wild-type levels in exo1∆ mutants (Figs. 2A andS2F). Finally, to understand how and when it functions, we assessed the role of Cdc5 polo kinase, required for meiotic crossover, in recruiting Mlh3 to recombination sites. We found that in a ndt80∆ mutant, where Cdc5 is not expressed, Mlh3 was still recruited at DSB sites (Figs. 2A andS2G) and also formed foci on pachytene chromosomes (Fig. 1J). We conclude that the lack of dHJ resolution in the absence of Cdc5 is not due to a failure to recruit Mlh1-3 to recombination sites, but possibly a failure to activate Mlh1-3 once recruited.

Having determined that Mlh3 binds at a late step of recombination, depending on ZMM proteins, makes it a good candidate for being a marker for crossovers, as in other organisms.

Mlh3 binding levels correlate well with DSB frequencies, except in centromeric and late replicating regions

Whether Mlh1-3-dependent crossover has regional specificity across chromosomal territories is an important question, knowing that crossovers mislocalization can lead to aneuploidies [START_REF] Lamb | Characterization of susceptible chiasma configurations that increase the risk for maternal nondisjunction of chromosome 21[END_REF]. To address this, we mapped Mlh3 binding sites by ChIP-seq from highly synchronized meiotic cultures thanks to a copper-inducible pCUP1-IME1 construct [START_REF] Chia | Temporal Expression of a Master Regulator Drives Synchronous Sporulation in Budding Yeast[END_REF]. We used the 5 and 5.5 h time-points, when crossovers appear (Fig. S3). We also mapped DSB sites by sequencing Spo11 oligonucleotides from pCUP1-IME1 synchronized cultures, at 5 h when DSB levels are maximal (Fig. S3). The Mlh3 binding map was highly similar to that of the Mer3 and Zip4 ZMMs: Mlh3 formed peaks around DSB hotspots, in addition to being weakly associated with axis binding sites (Figs. 2B, 2C, S4A and S4B).

To get insight into the mode of action of Mlh1-3 once on recombination intermediates, we next asked which DNA region Mlh3 occupies on individual DSB hotspots. Mlh3 peaks at DSB hotspots had a width (2.2 kb) similar to that previously determined for the length of resection tracts at DSB hotspots (1.6 kb) (39) (Fig. S4C), which is predicted to result in recombination intermediates of roughly similar size [START_REF] Marsolier-Kergoat | Mechanistic View and Genetic Control of DNA Recombination during Meiosis[END_REF]. Furthermore, in an exo1-D173A (exo1-nd) catalytic mutant that shows a 2.2 fold reduction in resection tracts lengths [START_REF] Zakharyevich | Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions[END_REF][START_REF] Mimitou | A global view of meiotic double-strand break end resection[END_REF], Mlh3 peaks at DSB hotspots were clearly less wide than in wild-type (Figs. S4C andS4D), consistent with the detectable Mlh1-3 distribution in vivo being mostly defined by the limits of recombination intermediates.

Among the 1155 Mlh3 peaks identified (Table S1), 484 overlapped at least one of the strongest 1000 DSB hotspots (Table S2), and among the strongest 200 DSB hotspots, 164 had a detected Mlh3 peak. In addition, the signal intensities of DSB and Mlh3 at the DSB hotspots were highly correlated (Fig. S4E). Despite this high global correlation between DSB and Mlh3 binding frequencies, we observed a significant reduction of Mlh3 binding per DSB in pericentromeric regions, (Figs.

2D and 2E and Figs. S5A andS5B) . By contrast, the ZMM Mer3 and Zip4 were not reduced at pericentromeric regions, but were reduced in subtelomeric regions (Figs. 2D andS5A), indicating a differential regulation than Mlh3.

Finally, we asked if the binding of ZMM and Mlh3 on DSB hotspots is modulated by replication timing. Whereas ZMM binding was not affected, Mlh3 binding per DSB was reduced in late replicating regions (Fig. 2F); this was seen in samples from both 5 h (Fig. S5C) and 5.5 h (Fig. 2F) suggesting that this effect is not due to temporal differences in Mlh3 loading between early and late replicating regions. Altogether, our results indicate a regional control of Mlh3 binding to DSBs, that operates negatively in centromeric and in late replicating regions.

MutLγ forms a complex with Exo1 and interacts with MutSγ in vivo

To further gain a mechanistic handle on the function of key players in meiotic crossover, we examined the in vivo interacting partners of MutLγ. We immunoprecipitated Mlh3 4 h after initiating meiosis, when crossovers appear (5), followed by proteomics mass spectrometry analysis (Fig. 3A). Mlh3 pulled down its MutLγ partner Mlh1, as expected, but also Exo1, providing in vivo support for previous proposals that it assists Mlh1-3 in crossover resolution [START_REF] Zakharyevich | Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions[END_REF] (Fig. 3A and Table S3). We confirmed this interaction by Western blot analysis of the reverse pull down of Mlh3 by Exo1 (Fig. 3B). Interestingly, Exo1 pulled down Mlh1 and Mlh3 throughout meiosis, and is therefore a constitutive partner of MutLγ (Fig. 3C). This interaction required the Mlh1 E682 residue that mediates the direct interaction between Exo1 and Mlh1 (Fig. 3B), implying that the interaction between Exo1 and MutLγ is mediated by Mlh1. Consistent with these results, an mlh1E682A mutant displays meiotic crossover defects [START_REF] Zakharyevich | Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions[END_REF]. We noticed that the Mlh1E682A protein is reproducibly present at lower levels than the wild-type Mlh1 protein, although previous studies showed that it is still proficient for mismatch repair. This may be due to its loss of interaction with several partners (Exo1, but also Sgs1, Ntg2) [START_REF] Dherin | Characterization of a highly conserved binding site of Mlh1 required for exonuclease I-dependent mismatch repair[END_REF].

Mlh3 immunoprecipitates also contained Pms1, the partner of Mlh1 in the MutL heterodimer (Fig. 3A, Table S3), and we confirmed this interaction in reverse Pms1 pulldowns (Fig. 3D). This possible MutL-MutLγ interaction prompted us to ask if MutLγ-MutLγ interactions also occur, possibly as a result of the proposed MutLγ polymerization needed to cleave DNA in vitro [START_REF] Manhart | The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans[END_REF]. However, we failed to detect any Mlh3-Mlh3 interaction in coimmunoprecipitation assays (Fig. 3D, lower panel and S5), suggesting either that MutLγ does not interact with itself in vivo or that this may be regulated and occur only at the time of CO formation, making it difficult to detect. Surprisingly, we did not recover any peptides from the Msh4-Msh5 (MutSγ) heterodimer or from other ZMM proteins in the Mlh3 immunoprecipitates (Table S3). A weak coimmunoprecipitation of Mlh1 and Mlh3 by Msh4 was detected, but the fraction of Mlh1 and Mlh3 was very low (Fig. 3B), consistent with the lack of visible co-localization of Mlh3 and Msh4 foci. This Mlh3-Msh4 interaction, even transient in vivo, supports recent in vitro data showing that the activity of the human MutLγ complex is strongly stimulated by both Exo1 and MutSγ [START_REF] Cannavo | Regulation of the MLH1-MLH3 endonuclease in meiosis[END_REF][START_REF] Kulkarni | PCNA activates the MutLgamma endonuclease to promote meiotic crossing over[END_REF].

Cdc5 kinase interacts with both MutLγ and Exo1 bound to recombination sites

We have shown that Cdc5 is not required for Mlh3 binding to DSB hotspots, and moreover its targets relevant for crossover formation by the Mlh1-Mlh3 pathway are still unknown [START_REF] Sourirajan | Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis[END_REF]. We did not detect Cdc5 in our meiotic Mlh3 pulldowns (Table S3), but to address this issue, we examined if Cdc5 interacts with MutLγ and Exo1 in highly synchronous meiotic cells. At the time when crossovers start to appear (5.5 h, Fig. S3), Cdc5 robustly immunoprecipitated Mlh1, Mlh3 and Exo1 (Fig. 4A), raising the possibility that it may directly activate MutLγ. Indeed, Cdc5 associated with recombination sites during meiosis, at the same time as Mlh3 (Fig. 4B). Since MutLγ associates with recombination sites in ndt80∆ mutants, where Cdc5 is absent (Fig. 2A), we propose that Cdc5 is recruited to a pre-existing Exo1-MutLγ complex bound at CO sites.

To know which of the MutL-Exo1 components is a direct target of Cdc5, we assessed their interdependency for in vivo coimmunoprecipitation with Cdc5.

Strikingly, Cdc5 still associated at normal levels with Mlh1 and Mlh3 in the absence of Exo1 (Fig. 4C), suggesting that MutLγ itself or another of its interacting partners may be a phosphorylation target of Cdc5. Moreover, Exo1 still coimmunoprecipitated with Cdc5 in the absence of interaction with MutLγ (Fig. 4D, mlh1E682A mutant, where Exo1 interaction with Mlh1-3 is abrogated), demonstrating that Cdc5 also interacts with Exo1.

Cdc5 directly interacts with Exo1 to promote crossover formation

We tested whether Cdc5 interacts directly with the above factors. Using a yeast two-hybrid assay, we found that the Cdc5 polo-box domain (PBD), known to be involved in interaction with Cdc5 targets, did interact with Exo1, whereas it did not with either Mlh1 or Mlh3 (Fig. S7A). Cdc5 PBD usually binds a priming phosphate, often formed by CDK1, in the context of a consensus motif, S-S(Phos)/T(Phos)-P [START_REF] Cheng | The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex[END_REF][START_REF] Elia | The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain[END_REF]. However, recombinant purified Exo1 strongly interacted with purified Cdc5 (Figs. 4E andS7B), even after pre-treating Exo1 with phosphatase, ruling out that this direct interaction is mediated through Exo1 phosphorylation.

A few other cases of phosphorylation-independent Cdc5 interactions have been described, including that between Cdc5 and Dbf4 [START_REF] Archambault | Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis[END_REF][START_REF] Bonner | Binding of Drosophila Polo kinase to its regulator Matrimony is noncanonical and involves two separate functional domains[END_REF][START_REF] Chen | Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction[END_REF]. Domain mapping defined a small region of Exo1 (531 to 591) necessary and sufficient for interaction with Cdc5 PBD using two-hybrid assays (Figs. 5A andS7C). This region contains a motif (570-575) conserved among yeast species, resembling the RSIEGA motif found in Dbf4 (Fig. 5B) [START_REF] Chen | Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction[END_REF]. The Cdc5 PBD structure has recently been solved, and shows a specific binding surface for the Dbf4 motif, opposite from the canonical phosphopeptide binding site (Fig. 5Ci) [START_REF] Almawi | Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division[END_REF]. Interestingly, the Exo1 RSIEGA-like motif could be modeled interacting exactly with the same Cdc5 surface as Dbf4 (Fig. 5Cii). Importantly, mutation of the key residue of this surface (S630Q mutation) abolished interaction with Exo1, as it did for Dbf4, but kept intact the interaction with Spc72, a canonical interacting substrate of Cdc5 (Figs.

5D and S7D) [START_REF] Almawi | Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division[END_REF]. Conversely, mutation of the WHK motif of Cdc5 involved in phosphopeptide recognition [START_REF] Elia | The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain[END_REF] did not alter interaction with Exo1 or Dbf4, but reduced interaction with Spc72 (Figs. 5D andS7D) [START_REF] Chen | Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction[END_REF][START_REF] Almawi | Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division[END_REF]. Furthermore, mutation of the equivalent of the key residues of Dbf4 of this motif in Exo1 (R570E I572D G574A, hereafter called exo1-cid, Cdc5 interaction-deficient) abolished the two-hybrid interaction between Exo1 and Cdc5 (Figs. 5A andS7C), confirming that Exo1 and Dbf4 bind in a similar way to Cdc5.

Interaction between Exo1-cid and Cdc5 was also strongly reduced in in vivo coimmunoprecipitation assays, to around 35% of wild type levels (Fig. 6A). In addition, exo1-cid mutants showed decreased crossovers, on two different tested chromosomes, at the HIS4LEU2 hotspot on chromosome III (Fig. 6B), and in the CEN8-ARG4 interval on chromosome VIII (Fig. 6C). Moreover, crossovers were especially reduced by the exo1-cid mutation combined with a triple nuclease mutant where only MutLγ-dependent crossovers remain (Figure 6B, mms4-md yen1∆ slx4∆, (9)), or with the single mms4-md mutant (Fig. 6C). In all cases examined, crossover levels in exo1-cid were greater than in the exo1∆ mutant, but decreased to the same extent as in mutants that impair Exo1 interaction with MutLγ (exo1-FF477AA and mlh1E682A mutants ( 19)), highlighting that the in vivo crossover function of Exo1 is lost in the exo1-cid mutant. To ensure that the crossover defects of Exo1-cid are due to its loss of interaction with Cdc5, we asked if forced recruitment of Cdc5 to this mutant protein restores crossovers. We therefore built strains where one copy of exo1-cid is fused to CDC5, behind the endogenous EXO1 promoter. To avoid confounding effects of expressing CDC5 at the same time as EXO1, and therefore throughout meiosis, we made a control where CDC5 was expressed from the EXO1 promoter without fusion to EXO1 (Fig. 6D). Furthermore, to avoid premature activation of the structure-specific nucleases due to early expression of CDC5 [START_REF] Matos | Regulatory control of the resolution of DNA recombination intermediates during meiosis and mitosis[END_REF][START_REF] Arter | Regulated Crossing-Over Requires Inactivation of Yen1/GEN1 Resolvase during Meiotic Prophase I[END_REF], we conducted the experiment in the triple nuclease background. Remarkably, only the fusion of Cdc5, but not its kinase-dead version, to Exo1-cid increased crossovers, to levels approaching wild-type Exo1 (Fig. 6D). Our results therefore demonstrate the direct function of Exo1 in recruiting Cdc5, for downstream phosphorylation of targets relevant for crossover.

Since Exo1 directly interacts with Cdc5, we assessed if Exo1 phosphorylation is important for crossovers. We mapped the in vivo Exo1 phosphorylation sites by mass spectrometry of Exo1-TAP purified from meiotic cells after induction of Cdc5, and identified S664 (within the CDK consensus SSP motif of Exo1) as being phosphorylated, along with eight other sites, including three previously described as resulting from DNA damage checkpoint activation (50) (Figs. S8A andB).

However, mutation of all nine phosphorylated residues of Exo1 (mutant exo1-9A) did not affect crossovers (Fig. S8C). These results converge to suggest that Cdc5 does not phosphorylate Exo1 itself, or if it does, it is not important for crossover formation. Instead, our data suggest that Exo1 interacts with Cdc5, to activate in situ the kinase activity of Cdc5 towards its targets, MutLγ or its partners.

Discussion

In this study, we provide the first thorough in vivo characterization of MutLγ complex function in meiotic crossovers. We show that MutLγ forms foci on meiotic chromosomes and that globally, its binding to recombination sites is repressed close to centromeres and in late replicating regions. This predicts that the outcome of meiotic recombination is specifically modulated at specific chromosomal regions. Relevant to MutLγ activation, we show a weak, possibly very transient physical interaction with MutSγ during meiosis and a robust, constitutive interaction with Exo1. We reveal a new non-catalytic function of Exo1 in interacting with Cdc5 to activate crossover formation. Our data highlight a noncanonical, direct interaction, which does not seem to be accompanied by Cdc5mediated Exo1 phosphorylation. We propose that Exo1 serves as a matchmaker and that direct binding of Cdc5 to Exo1 allows Cdc5 to be active and locally phosphorylate MutLγ (Fig. 7).

The map of Mlh3 binding sites is highly correlated with that of DSBs except in specific chromosome regions

The choice to make a crossover during meiotic DSB repair may be modulated along chromosomes, but a precise and high resolution comparison of DSB and crossover levels is missing (reviewed in ( 51)). In mice, studies of a few hotspots suggest that crossover/noncrossover ratios can vary from one hotspot to another [START_REF] Cole | Comprehensive, fine-scale dissection of homologous recombination outcomes at a hot spot in mouse meiosis[END_REF][START_REF] De Boer | The diverse roles of transverse filaments of synaptonemal complexes in meiosis[END_REF][START_REF] De Boer | Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hot spots in mice[END_REF][START_REF] Sarbajna | A major recombination hotspot in the XqYq pseudoautosomal region gives new insight into processing of human gene conversion events[END_REF]. Recent data comparing a genome-wide DSB map with existing genetic maps have shown that in female mice, crossover formation is repressed at subtelomeric regions [START_REF] Brick | Extensive sex differences at the initiation of genetic recombination[END_REF]. Improper placement of crossovers in the vicinity of centromeres is infrequent and negatively influences meiotic chromosome segregation [START_REF] Lamb | Characterization of susceptible chiasma configurations that increase the risk for maternal nondisjunction of chromosome 21[END_REF][START_REF] Kuhl | Kinetochores, cohesin, and DNA breaks: Controlling meiotic recombination within pericentromeres[END_REF][START_REF] Rockmill | Centromere-proximal crossovers are associated with precocious separation of sister chromatids during meiosis in Saccharomyces cerevisiae[END_REF]. Although recombination close to centromeres is already reduced at the level of DSB formation, residual DSBs increase the risk of aneuploidy [START_REF] Pan | A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation[END_REF]. One way to prevent crossover formation by pericentromeric DSBs is to repair them from the sister chromatid and/or as noncrossovers [START_REF] Chen | Global analysis of the meiotic crossover landscape[END_REF][START_REF] Vincenten | The kinetochore prevents centromere-proximal crossover recombination during meiosis[END_REF]. We found that Mlh3/DSB ratios are lower in pericentromeric regions than along chromosome arms, which may also contribute to the lower crossover levels per DSB observed in pericentromeres. Since ZMM binding is not significantly reduced, we propose that there is a deselection of ZMM-bound at pericentromeric recombination sites, which are then preferentially repaired without MutL binding, as noncrossovers. In this regard, the phosphatase PP2A associated to Shugoshin has been shown to counteract cohesin kinases, including Cdc5, in pericentromeric regions [START_REF] Galander | Spo13 prevents premature cohesin cleavage during meiosis[END_REF]. It may also prevent Cdc5-driven MutL activation in these regions.

Prior to our study, it was not clear if replication timing also influenced the choice to repair DSBs as a crossover [START_REF] Borde | Direct coupling between meiotic DNA replication and recombination initiation[END_REF][START_REF] Buhler | Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae[END_REF]. The fact that Mlh3 binding per DSB is less frequent in late replicating regions suggests there may be a preferred time window for loading MutL onto recombination intermediates, such that DSBs occurring late as a consequence of late replication would be less likely to load MutL. It remains to be determined if these DSBs are repaired by the other nucleases able to generate crossovers (such as Mus81-Mms4) or if they are repaired through the noncrossover pathway. Recent cytological data in tomato suggested that the different crossover promoting nucleases (MutLγ and the structure-specific nucleases) are differently distributed along chromosomes [START_REF] Anderson | Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers[END_REF].

Mlh3 associates with DSB sites, at a late step of recombination, and forms foci distinct from ZMM-bound foci

MutLγ is predicted to act on recombination intermediates that have been stabilized by ZMM proteins, consistent with our finding that, in their absence, Mlh3 binding is absent (zip3∆ and mer3∆) or reduced (msh4∆). The fact that we did not see colocalization between ZMM and Mlh3 foci suggests that they are two temporally distinct species that form on recombination intermediates, at least as visible entities. Indeed, we detect much less (16 on average) Mlh3 foci than the estimated Mlh1-Mlh3 dependent crossovers (around 80). Zip3 and Msh4 foci would precede Mlh3 foci, to preserve the dHJs and hand them over to MutLγ, thereby positioning and activating MutLγ on the recombination intermediates, without staying on chromatin as visible foci when the Mlh3 focus forms (Fig. 7).

Our results are consistent with those in mice, where early co-localization between Msh4 and Mlh1 foci disappears at mid-pachytene [START_REF] Kneitz | MutS homolog 4 localization to meiotic chromosomes is required for chromosome pairing during meiosis in male and female mice[END_REF][START_REF] Santucci-Darmanin | The DNA mismatch-repair MLH3 protein interacts with MSH4 in meiotic cells, supporting a role for this MutL homolog in mammalian meiotic recombination[END_REF].

In budding yeast, ZMM foci (around 45 per nucleus) are proposed to all give rise to crossovers (around 75 ZMM-dependent crossovers per meiosis) [START_REF] Serrentino | The spatial regulation of meiotic recombination hotspots: Are all DSB hotspots crossover hotspots?[END_REF][START_REF] Mancera | Highresolution mapping of meiotic crossovers and non-crossovers in yeast[END_REF], contrary to plants, mammals and Sordaria where only a subset of ZMM foci result in crossovers [START_REF] Chelysheva | The Arabidopsis HEI10 Is a New ZMM Protein Related to Zip3[END_REF][START_REF] Muyt | E3 ligase Hei10: a multifaceted structure-based signaling molecule with roles within and beyond meiosis[END_REF][START_REF] Reynolds | RNF212 is a dosage-sensitive regulator of crossing-over during mammalian meiosis[END_REF][START_REF] Sun | Transcription dynamically patterns the meiotic chromosomeaxis interface[END_REF]. However, the fact that we detect less Mlh3 foci (16 per nucleus) than ZMM suggests that a similar selection of a subset of ZMM-bound sites may operate in budding yeast, to become bound by MutLγ, the remaining being dismantled by helicases into noncrossover products. In Sordaria, Msh4 foci numbers diminish between early and mid-pachytene, a time frame in which Mlh1-Mlh3 is believed to be recruited [START_REF] Storlazzi | Recombination proteins mediate meiotic spatial chromosome organization and pairing[END_REF]. An alternative hypothesis is that Mlh3 residence time on late crossover intermediates may be limited and therefore Mlh3 is not detected as a focus within a single nucleus on all crossover sites. Finally, we cannot exclude that the foci we detect, which are much fewer (16 on average) than predicted MutLγ-dependent crossovers [START_REF] Zalevsky | Crossing Over During Caenorhabditis elegans Meiosis Requires a Conserved MutS-Based Pathway That Is Partially Dispensable in Budding Yeast[END_REF], represent only a subset (unproductive, or at a particular recombination stage) of all the Mlh3 bound sites.

MutLγ forms a constitutive complex with Exo1 in meiotic cells but interacts only transiently with MutSγ

In many organisms (budding yeast, mice and plants) msh4 or msh5 mutants have an earlier meiotic defect than MutLγ mutants, but it is attractive to propose, by analogy to mismatch repair, that MutSγ also works directly with MutLγ-Exo1 for crossover formation. Consistent with an early role for MutSγ, we show that most Msh4 foci do not colocalize with Mlh3, but that Msh4 is required for the association of Mlh3 with recombination sites. In addition, the detected interaction between a small fraction of MutSγ with MutLγ in yeast meiotic cells fits also with a late function for MutS through direct activation of MutLγ. A similar interaction was also described for the cognate mouse proteins [START_REF] Santucci-Darmanin | The DNA mismatch-repair MLH3 protein interacts with MSH4 in meiotic cells, supporting a role for this MutL homolog in mammalian meiotic recombination[END_REF]. A recent study reports that human MutS strongly stimulates human MutLγ endonuclease in vitro [START_REF] Cannavo | Regulation of the MLH1-MLH3 endonuclease in meiosis[END_REF].

Together with our cytological and interaction data, we can then propose that in vivo, MutSγ contributes by creating the proper substrate where the MutLγ-Exo1 ensemble loads, and that a subset of MutSγ may subsequently activate MutLγ catalysis by direct interaction with MutLγ (Fig. 7). However, it is worth noting that, in C. elegans, MutSγ promotes crossovers and remains bound to crossover sites, although crossover resolution is not achieved by MutLγ, suggesting that, in this organism, MutSγ cooperates, directly or indirectly, with a nuclease other than MutLγ [START_REF] Woglar | Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination[END_REF][START_REF] Zalevsky | Crossing Over During Caenorhabditis elegans Meiosis Requires a Conserved MutS-Based Pathway That Is Partially Dispensable in Budding Yeast[END_REF]. Further work aiming at genetically separating these two functions of MutSγ will clarify these issues.

Another example of non-canonical Cdc5 binding to its substrates, involving the RSIEGA motif

We found that Exo1 interacts with Cdc5 through the same mode as the only example described so far, Dbf4. Besides the importance for the meiotic recombination context, our study therefore adds another case where the recently described interaction surface on Cdc5, distinct from the phosphopeptide recognition surface, promotes interaction with a protein relevant for a biological process [START_REF] Almawi | Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division[END_REF]. This surface is conserved in human PLK, and our work should open new avenues to search for other partners of Cdc5 or human PLKs with similar interacting motifs, and to inhibitors that may specifically target this new interacting surface.

Exo1 serves as a Cdc5 recruiting/activating platform for MutLγ crossover formation

Our study is the first to establish a functional molecular link between Cdc5 and the MutLγ-Exo1 complex, providing some explanation for why this kinase is important for meiotic crossovers [START_REF] Sourirajan | Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis[END_REF]. We show that Exo1 is constitutively associated with MutLγ, and therefore the binding of Exo1 to MutLγ is not sufficient in vivo to activate resolution. Rather, our data, in particular the observation that mutants blocking Exo1-Cdc5 interaction have crossover defects, suggest that resolution is activated by Cdc5 through its direct interaction with Exo1. What could be the downstream event triggered by Cdc5 binding to Exo1? Cdc5 function in MutL crossovers involves phosphorylation of at least a substrate, because induction of a kinase-dead Cdc5 in meiosis is not sufficient to promote crossover formation [START_REF] Sourirajan | Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis[END_REF] and because the forced recruitment of Cdc5 to Exo1 only stimulates crossover when Cdc5 kinase activity is intact (our results).

It is unlikely that Exo1 is the relevant phosphotarget, since mutation of Exo1's meiotic phosphorylated residues that we have detected had no effect on crossovers. Another obvious candidate for phosphorylation, once Cdc5 binds Exo1, is the MutLγ complex itself. However, attempts failed to detect Cdc5 activation-associated change in the mobility of Mlh1 or Mlh3 (this study, Fig. 3C, and ( 76)), and published meiotic phosphoproteomes datasets do not contain meiotic phosphorylation sites on Mlh1 or Mlh3 that would appear upon Cdc5 induction [START_REF] Suhandynata | Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics[END_REF]. Another possible target is the Chd1 protein, recently described as a binding partner of Exo1 and important for MutLγ crossovers [START_REF] Wild | Network Rewiring of Homologous Recombination Enzymes during Mitotic Proliferation and Meiosis[END_REF]. A proposed function of this chromatin remodeler is to allow MutLγ polymerization along DNA, an event suggested important for in vitro nuclease activity on naked DNA [START_REF] Manhart | The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans[END_REF][START_REF] Wild | Network Rewiring of Homologous Recombination Enzymes during Mitotic Proliferation and Meiosis[END_REF] and that would require chromatin remodeling in vivo. Although we failed at detecting indications that MutLγ polymerizes (no MutLγ-MutLγ interaction, no large spreading of Mlh3 peaks around DSB hotspots), we cannot exclude that once loaded, MutLγ may polymerize transiently when becoming active on a region broader than recombination intermediates (Fig. 7). Further experiments examining Mlh3 dynamics at the time of dHJ resolution and the influence of Cdc5 and Chd1 will be required to examine this possibility.

It is noteworthy that despite the reduced crossovers seen when Cdc5 no longer directly interacts with Exo1, Cdc5 is able to coprecipitate with MutLγ from meiotic cells even in the absence of Exo1, and that the Exo1-cid mutant, although totally deficient for two-hybrid interaction with Cdc5, also shows residual coprecipitation with Cdc5 in vivo, likely via its interaction with MutLγ. Therefore, rather than being essential to bring Cdc5 to the MutLγ-Exo1 complex, direct Exo1 binding may serve as the molecular switch to allow Cdc5 phosphorylate its targets (Fig. 7).

As a whole, our study reveals that MutLγ activation is tightly controlled in vivo, both locally through the direct coupling of crossover formation with key meiotic progression steps by the Cdc5 kinase, and globally by the underlying chromosome structure. Much of this regulation is likely to operate in mammals, where the key proteins are conserved. Our study provides an illustration of how nucleases at risk of impairing genome integrity in the germline are kept in check through multiple control levels.

Materials and methods

Yeast manipulations

All yeast strains are derivatives of the SK1 background except those used for 2 hybrid and mutation analyses (Table S4), and the strains used in specific figures are listed in Table S5. All experiments were performed at 30°C. Growth conditions and strain constructions are described in Supplemental Methods

Analysis of crossover frequencies

For crossover analysis at HIS4LEU2, cells were harvested from meiotic time courses at the indicated time point. 2 μg genomic DNA was digested with XhoI and analyzed by Southern blot using a labeled DNA probe A as described [START_REF] Hunter | The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination[END_REF]. The radioactive signal was detected by a Phosphorimager (Typhoon, GE Healthcare) and quantified using the Image Quant software as described [START_REF] Duroc | Concerted action of the MutLbeta heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion[END_REF]. For genetic distances on chromosome VIII, diploids were sporulated in liquid medium, and recombination between fluorescent markers was scored after 24h in sporulation, by microscopy analysis as described [START_REF] Thacker | Exploiting spore-autonomous fluorescent protein expression to quantify meiotic chromosome behaviors in Saccharomyces cerevisiae[END_REF]. Two independent sets of each strain were combined and at least 600 tetrads were counted. Genetic distances in the CEN8-ARG4 interval were calculated from the distribution of parental ditype (PD), nonparental ditype (NPD) and tetratype (T) tetrads and genetic distances (cM) were calculated using the Perkins equation: cM = (100 (6NPD + T))/(2(PD + NPD + T)). Standard errors of genetic distances were calculated using Stahl Lab Online Tools (https://elizabethhousworth.com/StahlLabOnlineTools/). Flag-affinity pull-down, proteomics mass spectrometry and coimmunoprecipitation analyses 2.10 10 cells (for Flag pull-down and mass spectrometry analysis) or 1.2 10 9 cells were harvested from synchronous meiotic cultures at the indicated time after meiosis induction. Details are provided in the Supplemental Methods.

Recombinant proteins and interaction assays

Recombinant yeast Exo1-Flag and Cdc5 were purified from insect cells. Details of protein purification and Exo1-Flag pull-downs are in the Supplemental Methods.

Yeast two-hybrid assays

Yeast two-hybrid assays were performed exactly as described [START_REF] Duroc | Concerted action of the MutLbeta heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion[END_REF]. Cloning details are in the Supplemental Methods.

Modeling of Dbf4 and Exo1 peptides on Cdc5 PBD structure

The RSIEGA motif of Exo1 was modeled based the structure of Cdc5 bound to a Dbf4-derived peptide encompassing the RSIEGA motif of Dbf4 (PDB ID: 6MF6; [START_REF] Almawi | Distinct surfaces on Cdc5/PLK Polo-box domain orchestrate combinatorial substrate recognition during cell division[END_REF]. The sequence was edited in Coot [START_REF] Emsley | Coot: model-building tools for molecular graphics[END_REF] and the rotamers of each residue chosen to prevent clashes between Cdc5 and the modeled peptide. Cytology 4x10 7 cells were harvested at the indicated time-point and yeast chromosome spreads were prepared as described [START_REF] Xaver | Smc5/6-Mms21 prevents and eliminates inappropriate recombination intermediates in meiosis[END_REF]. Mlh3-myc18 was stained with primary anti-myc rabbit antibody (Abcam, 1:600) and secondary FITC-conjugated antirabbit (Thermo Fischer 1:600) for all cytological experiments except for the colocalization with Zip3, for which primary 9E11 anti-myc mouse antibody was used together with a secondary Cy3-conjugated anti-mouse (Jackson ImmunoResearch, 1:600). Msh4-HA3 was stained with primary anti-HA mouse antibody (Anopoli, 1:1000) and secondary Cy3-conjugated anti-mouse antibody (Thermo Fischer 1:600) when used in parallel with rabbit based anti-myc. Zip3 was stained with a primary anti-Zip3 rabbit antibody (1:2000), a gift from Akira Shinohara. The secondary antibody was Cy5-conjugated anti-rabbit (Amersham, 1:500). Fluorescent microscopy was carried out on a ZEISS AXIO Imager M2 with a ZEISS Plan-Neofluar 100x, and a 2x additional magnification by a Zeiss optovar.

Images were taken at an exposure of 1 second for DAPI (BFP channel), Cy3 (Cy3 channel), FITC (FITC channel) and 2 seconds for Cy5 (AF660 channel). The Light source: Sola SM II (Llumencor); camera: CoolSNAP HQ2 (Visitron Systems GmbH); acquisition software: Visiview (Visitron Systems GmbH). Nuclei acquired with this setup were analyzed by Fiji software and R-scripts.

Chromatin immunoprecipitation, real-time quantitative PCR and ChIP-seq

For each meiotic time point, 2.10 8 cells were processed as described [START_REF] Duroc | Concerted action of the MutLbeta heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion[END_REF]. Details are in the Supplemental Methods

Spo11 oligonucleotide mapping

Spo11-Flag oligonucleotides were purified and processed for sequencing library preparation and analyzed as previously described [START_REF] Lam | Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast[END_REF], from synchronous pCUP1-IME1 strains (see Supplemental Methods).

Sequencing data processing and bioinformatic analyses

ChIP-seq and Spo11 oligo data were analyzed and normalized mostly as described [START_REF] Muyt | A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation[END_REF][START_REF] Lam | Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast[END_REF]. Details are provided in the Supplemental Methods.

Data availability

Sequencing data were deposited at the Gene Expression Omnibus (GEO) with the accession numbers GSE133108 (Spo11 oligos in pCUP1-IME1), GSE132850 (Mer3 and Mlh3 ChIP-seq). Proteomic data were deposited on the Proteome Xchange with the identifiers PXD014180 (Mlh3 pulldowns) and PXD014185 (Exo1 phosphosites).

All data are included in the article and SI Appendix (Table S6). : mass-spectrometry analysis of selected proteins reproducibly identified in all replicates and not in the controls (no tag strain). The number of peptides of 4 independent experiments is shown (two with benzonase treatment, two without). Detail of pulled-down proteins in Table S3. MutSγ stabilizes dHJ intermediates and forms foci on chromosomes. MutLγ, in complex with Exo1, binds dJH intermediates that have been stabilized by MutSγ and other ZMM proteins. For simplicity, MutSγ is shown embracing both DNA duplexes of the intermediates, but recent data also suggest it may embrace separately each DNA duplex [START_REF] Woglar | Dynamic Architecture of DNA Repair Complexes and the Synaptonemal Complex at Sites of Meiotic Recombination[END_REF]. Once bound on dHJ, MutLγ may form a focus when interacting with MutSγ (model shown here), or later, in the process of its activation (not shown). Upon NDT80 activation, the Cdc5 kinase is induced, and interacts with the MutLγ-Exo1 complex through multiple interactions (green cloud). Among these interactions, the direct interaction of Cdc5 with Exo1 is important for MutLγ-driven crossover formation. We propose that this interaction allows Cdc5 to phosphorylate MutLγ, which activates its nuclease function and produces crossover formation. This may occur through transient MutLγ polymerization.

  ). Importantly, Mlh3 foci were largely absent in zip3∆ and in substantially reduced numbers in msh4∆ mutants (Figs. 1F, 1G and 1H), in agreement with genetic data showing that MutLγ acts downstream of ZMM proteins. Surprisingly, although Zip3 and to a lesser extent Msh4 are important for Mlh3 foci, Mlh3 showed very little colocalization with either Zip3 or Msh4 (Figs. 1B, 1C and 1I), suggesting that Mlh3 coexists only briefly with the other two components at the same intermediates. We conclude that Zip3 and Msh4 ZMM are required for normal Mlh3 foci formation, and propose that Zip3 and Msh4 form earlier foci on recombination intermediates, before Mlh3 binding, which may reflect their function for stabilization of intermediates.

(

  C) Average ChIP-seq signal at the indicated features. Same data as in (B). The Mer3 and Zip4 ChIP-seq signals are aligned on the Spo11 hotspots midpoints from (82), and Mlh3 ChIP-seq signal on the pCUP1-IME1 Spo11 hotspots midpoints (this study). Lower panel, ChIP-seq signal is aligned on the Red1 peaks summits from (72). (D) ZMM and Mlh3 signals per DSB vary with the proximity to a centromere or a telomere. The ChIP-seq signal of each protein divided by their corresponding Spo11 oligo signal (Spo11 signal from (82) for Mer3 and Zip4, and pCUP1-IME1 Spo11 signal for Mlh3) was computed on the width plus 1 kb on each side of the strongest corresponding 2000 Spo11 hotspots at the indicated chromosome regions: interstitial (1805 and 1829 hotspots); 0 to10 kb from a centromere (29 and 28 hotspots); 10 to 20 kb from a centromere (50 and 46 hotspots); 0 to 40 kb from a telomere (117 and 97 hotspots). For each region, the corresponding interstitial control was computed by randomly selecting groups of interstitial hotspots regions with the same median Spo11 oligo level, this step was repeated 10000 times. Statistical differences (Mann-Whitney-Wilcoxon test) between different regions and their interstitial control are indicated. (E) Examples of DSB (pCUP1-IME1 Spo11 oligo) and Mlh3 binding in an interstitial region and two pericentromeric regions. The normalized signal smoothed with a 200 bp window size is indicated. (F) The ZMM and Mlh3 signals per DSB vary with the timing of DNA replication. Same legend as in (D) for the indicated chromosomal regions, except that early and late regions were directly compared to each other. See also Figs. S2, S3, S4 and S5.

Fig. 3 :

 3 Fig. 3: MutLγ forms a complex with Exo1 and transiently interacts with MutSγ in vivo (A) Affinity pull-down of Mlh3-Flag from cells at 4 h in meiosis. Silver-stained gel of pulled-down proteins.Table: mass-spectrometry analysis of selected proteins

(

  B) Coimmunoprecipitation by Exo1-TAP or Msh4-TAP from cells at 4 h in meiosis analyzed by Western blot. The asterisk indicates the uncleaved Msh4-TAP, of which the protein A region is weakly recognized by the anti-HA antibody. The Tev-cleaved Msh4-TAP is no longer recognized by the anti-HA. (C) Exo1-TAP pull-down throughout meiosis. The graph indicates the ratio of immunoprecipitated Mlh1 and Mlh3 relative to Exo1 in the Tev eluates at the indicated times in meiosis. Values are mean ± SD of two independent experiments. (D) Coimmunoprecipitation between Pms1-Flag and Mlh3-Myc (upper panel) or between Mlh3-Flag and Mlh3-Myc (lower panel) from cells at 4 h in meiosis analyzed by Western blot. Asterisks indicate a non-specific cross-hybridizing bands. See also Fig. S6.

Fig. 4 :

 4 Fig. 4: The Cdc5 kinase interacts with both MutLγ and Exo1 bound on recombination sites (A) Coimmunoprecipitation by Cdc5-TAP of Mlh1-HA, Mlh3-Myc and Exo1-Myc from pCUP1-IME1 synchronized cells at 5 h 30 in meiosis analyzed by Western blot. (B) Mlh3-Myc and Cdc5-TAP association with the indicated DSB hotspots as revealed by ChIP and qPCR at the indicated times of a pCUP1-IME1 synchronized meiotic time-course. Same normalization as in Fig. 2. Values are the average of three (Mlh3-Myc) or four (Cdc5-TAP) independent experiments ± S.E.M. (C) Coimmunoprecipitation by Cdc5-TAP of Mlh1-HA and Mlh3-Myc is independent of Exo1. Same conditions as in (A).

(

  D) Coimmunoprecipitation by Cdc5-TAP of Exo1 is independent of Exo1 interaction with MutLγ. Same conditions as in (A).

(

  E) Direct, phosphorylation-independent interaction between recombinant Exo1 and Cdc5 proteins. Western Blot showing the pulldown of purified Cdc5-PM (phosphomimetic, see Methods) by Exo1-Flag, in the presence or absence of CDK1 or lambda phosphatase. See also Fig. S7.

Fig. 5 :Fig. 7 :

 57 Fig. 5: Cdc5 directly interacts with Exo1 through a non-canonical site

Acknowledgments

We thank Wolf D. Heyer for helpful suggestions, Michael Lichten and Arnaud De Muyt for critical comments on the manuscript, Hajime Murakami for the meiotic replication data, Angelika Amon and Folkert van Werven for the pCUP1-IME1 construct, Hardeep Kaur and Michael Lichten for the pGAL1-CDC5 strain, Rajeev Kumar for constructing the Mlh3-Myc8 allele and Akira Shinohara for the anti-Zip3. We thank the Institut Curie PICT-IBISA Pasteur Imaging facility, member of the France Bioimaging National Infrastructure (ANR-10-INBS-04) and the NGS platform, supported by the grants ANR-10-EQPX-03 and ANR10-INBS-09-08 and by the Cancéropôle Ile-de-France. This work was supported by the Institut Curie and the CNRS; by Agence Nationale de la Recherche (ANR-15-CE11-0011) to V.B., J.-B.C. and P.C.; by the French Infrastructure FRISBI (ANR-10-INBS-05) to J.-B.C, and by "Région Ile-de-France" and the FRM medical grants to D.L. A.S. was supported by a postdoctoral fellowship from the Fondation ARC and by the Labex DEEP (ANR-11-LBX-0044).

Figure legends


Yujia Liu

Mana Kigure

Kyoka Koizumi

Nobuhiro Takeda

Masafumi Unno

Armelle Ouali

Synthesis of Tetrachloro, Tetraiodo, and Tetraazido Double-Decker Siloxanes

A convenient and scalable (gram-scale) route to unprecedented T8D2-DDSQs bearing four chloro-(3b) or four azido (5b) groups is reported. Both compounds were characterized and proved to undergo successfully nucleophilic substitution for 3b (with iodide or azide) and coppercatalyzed azide-alkyne [3+2] cycloaddition for 5b. All these transformations occurred under mild conditions and the corresponding DDSQs were prepared in very high yields. Beyond the enhanced multivalency as compared to the previously described di-substituted D2T8 structures, the reported tetra-functional DDSQs are formed as a single isomer and readily isolated in very high yields.

Moreover, the tetra-azido DDSQ 5b constitutes a multipurpose nano-building block for the further preparation of new inorganic-organic hybrid materials where the covalent incorporation of a DDSQ moiety brings valuable properties.

Introduction

Polyhedral oligomeric silsesquioxanes (POSSs), compounds with a rigid inorganic core composed of Si-O-Si bonds framework surrounded by organic groups, find increasing applications in various fields including polymers, 1 sensors, 2 catalysis, 3 host-guest complexes, 4 organic light-emitting diodes [START_REF] Walczak | Unusual cis and trans architecture of dihydrofunctional double-decker shaped silsesquioxane and synthesis of its ethyl bridged πconjugated arene derivatives[END_REF] or porous materials [START_REF] Liu | Selective dye adsorption and metal ion detection using multifunctional silsesquioxane-based tetraphenylethene-linked nanoporous polymers[END_REF] . [START_REF] Du | Cage-like Silsesquioxanes-based Hybrid Materials[END_REF] While a wide array of mono-or octafunctional derivatives of T8 silsesquioxanes have been described, 8 the double-decker siloxanes (DDSQs) bearing reactive substituents have been less studied (Scheme 1). Both open M4T8 (DDSQ-4OSi, Scheme 1a) and closed D2T8 (DDSQ-2OSi, Scheme 1b) frameworks are yet applied in various areas of chemistry such as coordination chemistry, 9 optics 10 or hybrid polymer nanocomposites 11 . [START_REF] Dudziec | Double-Decker Silsesquioxanes: Current Chemistry and Applications[END_REF] To include DDSQ architectures in more complex systems (for example coordination complexes, electronic or optic devices, hybrid polymers, and so on), the pre-requisite consists in decorating the M4T8 and D2T8-DDSQ architectures with reactive groups (Scheme 1a and 1b respectively). Therefore functionalizable versions were reported with M4T8 structures bearing for example four hydrogen, alkenyl or silylalkynyl groups, prone to undergo hydrosilylation or silylative couplings respectively (Scheme 1a). [START_REF] Dudziec | Double-Decker Silsesquioxanes: Current Chemistry and Applications[END_REF]13 In the same way, related D2T8 involving two reactive groups (one on each external D-type silicon atom) such as hydrogen, 14 alkenyl, 15 amino, 16 chloro 17 or azido [START_REF] Ervithayasuporn | Synthesis and Characterization of Highly Pure Azido-Functionalized Polyhedral Oligomeric Silsesquioxanes (POSS)[END_REF] were described as well (Scheme 1b).

Scheme 1. General structures of double-decker siloxanes (DDSQs) and reactive groups (RG) reported It is worthy to note that the number of reactive groups grafted on the inorganic core appears as decisive to define the further properties of the POSSs 13a as illustrated by the strongly different behaviours of mono-or polysubstituted POSS in a given application. For example, T8 bearing one boron-dipyrromethene fluorophore displayed significantly better lasing efficiency and photostability than their octafunctionalized counterparts while multivalency is a strong asset for catalytic or biological applications. 8 The fine tuning of external functional groups on the POSS thus constitutes a strategic challenge. Along these lines, we recently described the first tetrafunctional D2T8 DDSQs decorated with four vinyl-and allyl moieties (Scheme 1c). [START_REF] Liu | Characterization, and Functionalization of Tetrafunctional Double-Decker Siloxanes[END_REF] To open the way to new applications, the discovery of tetrafunctional DDSQs bearing key reactive groups such as azides is of great importance. Indeed the copper-catalyzed Huisgen [3+2] cycloaddition (CuAAC) involving an azide and an alkyne is highly attractive in terms of atom-economy, high yield and also for the robustness of the triazole ring it generates. 20 This ligation method is widely exploited to functionalize molecules or nano-objects for preparing functional materials. Therefore, azido-containing POSSs with several inorganic frameworks have been reported. Most examples involve T8 bearing one 21,22 or eight 23,24 azido terminal functions connected to the inorganic core through aliphatic or benzylic spacers. These cubic silsesquioxanes were functionalized via the CuAAC strategy to access to hybrid polymeric materials, 21,23 bio-active compounds, 24 or recyclable catalysts. 22 T10-POSSs and D2T8 DDSQs respectively decorated by ten or two azido substituents were significantly less explored although the preliminary reports demonstrate that they constitute interesting building blocks to design hybrid polymers 18b,25 or recyclable catalysts. 22c In this work, a synthetic route to an unprecedented tetraazido D2T8 DDSQ is described. Moreover, the proof of concept of its successful functionalization through CuAAC is provided to reach nitrogen-rich compounds prone to be of interest in several application fields. In addition, the described and characterized intermediate tetrachloro-and tetraiodo-DDSQs constitute appealing functionalizable building blocks for the preparation of organic-inorganic hybrid materials as well.

RESULTS AND DISCUSSION

Previously described tetravinyl 1a and tetraallyl DDSQ 1b were allowed to react with (chloromethyl)dimethylsilane 26 2 in the presence of Karstedt's catalyst (~ 0.02 mol% Pt per olefin group) and in both cases, the disappearance of the signals corresponding to olefinic signals was observed (Scheme 2). In the case of 1a, the quantitative functionalization of vinyl groups required a temperature of 40 °C while the hydrosilylation of 1b occurred selectively at 25 °C. The corresponding tetrachloro-DDSQ 3a was obtained in 81% yield after recrystallization and the pure compound 3b could be isolated in quantitative yield after a straightforward filtration through a silica and celite plug.

As expected, both 29 Si spectra of 3a and 3b clearly displayed two singlets for the non-equivalent T-unit silicon atoms of the core (Figure 1a and1b), the resonances at -79.60 and -79.10 ppm (respectively for 3a and 3b) being assigned to the internal T-group silicon atoms (Si in green) and those at -78.52 and -78.27 being assigned to the T-group silicon atoms (red color) nearest to the D-group silicon atoms (blue color). The latter gave rise to a singlet at around -19.4 ppm for both compounds and lastly, the 29 Si resonances at 5.36 and 3.39 ppm have been assigned to the silicon atoms of the carbosilane pattern (pink color) of 3a and 3b respectively. Moreover, the data obtained from 1 H and 13 C NMR were in accordance with the molecular structures (see supporting information). Then single crystals were grown and the structures of 3a and 3b could be determined by X-ray crystallography (Figures 2 and3, respectively). Both structures have similar framework, with Si-O-Si bond lengths in the ranges of 1.599(6)-1.639(6) Å (average 1.617(6) Å) for 3a and 1.598(0)-1.632(0) Å (average 1.615(0) Å) for 3b which are in accordance with previously reported related DDSQs derived from the 1a and 1b. [START_REF] Liu | Characterization, and Functionalization of Tetrafunctional Double-Decker Siloxanes[END_REF] Si-C lengths, in the ranges of 1.828-1.894 Å (average 1.858 Å) for 3a and of 1.760-1.980 Å (average 1.860 Å) for 3b were also consistent with those found for related compounds obtained after hydrosilylation with dimethylphenylsilane of 1a (in the ranges of 1.843-1.889 Å (average 1.858 Å)) or 1b (1.844-1.885 Å (average 1.861 Å)). [START_REF] Liu | Characterization, and Functionalization of Tetrafunctional Double-Decker Siloxanes[END_REF] Besides, in 3a, the Si-O-Si bond angles vary from 138.9 to 174. The chloro substituents of 3a and 3b could be quantitatively replaced by iodides through halide exchange in the presence of sodium iodide (Scheme 2). Resulting DDSQs 4a and 4b could be isolated in quantitative yields and fully characterized (multinuclear NMRs and mass spectroscopy, X-ray structures for 4a, see SI). These compounds represent useful building blocks for further functionalizations, iodides being easier to substitute as compared to parent chlorides.

It is worthy to note that when scaling up to the gram scale, the preparation of 3a through hydrosilylation of 1a with 2 occurred not selectively and the purification revealed difficult. Despite several attempts of purifications including column chromatography, gel permeation chromatography, washings and crystallization, 3a could not be obtained from minor impurities likely due to the partial polymerization of the C=C bonds. Therefore the following functionalizations were performed with the tetrachloro-DDSQ 3b, readily prepared in gram scale.

Next the chloro-decorated double-decker siloxane 3b was allowed to react with sodium azide in a THF/DMF mixture. The use of sub-stoichiometric amounts of sodium iodide greatly accelerated the nucleophilic substitution that proceeded more rapidly and selectively. Compound 3b yielded quantitatively and selectively to 5b, the first double-decker structure bearing four azido groups.

The 29 Si spectrum of 5b displayed the four expected singlets, analogous to those described in the cases of 3a and 3b (-79.08 and -78.25 ppm for the T silicon atoms of the core in green and red, respectively, -19.39 ppm for the D silicon atoms in blue and 3.18 ppm for the carbosilane in pink, Figure 1c). By 1 H NMR, the singlet assigned to the methylene group in the alpha position of the carbosilane (pink Si) next to the chloro-substituent in 3b was found to be affected to a very small extent by the substitution with the azido group (2.55 ppm in 3b to 2.50 ppm in 5b, see SI). However, by 13 C NMR, the signal of the same methylene group significantly shifted from 30.42 ppm in 3b to 40.95 ppm in 5b thus confirming the successful and quantitative substitution. These chemical shifts in 1 H and 13 C NMR are in accordance with those reported for related compounds bearing the Si-CH2-X moiety (X = Cl or N3). 27 The structure of 5b could be confirmed by 1 H and 13 C NMR as well. Moreover, the infra-red spectrum highlighted the characteristic band corresponding to the azido group at 2090 cm -1 . Lastly, crystals were grown and the structure of 5b could be determined by X-ray crystallography (Figure 4 As expected, the results of thermogravimetric analysis (TGA) under N2 showed a lower Td5 (167 °C, see SI Table S2) for azido DDSQ 5b as compared to those of tetra-chloro-DDSQs 3a and 3b (371 and 359 °C respectively) displaying a very high thermal stability.

Scheme 3. Functionalization of the four azido groups via copper-catalyzed [3+2] cycloaddition

The reactivity of the external azido substituents was next evaluated. For a representative example, 5b was allowed to react through copper-catalyzed Huisgen [3+2] cycloaddition with 3ethynylpyridine, a challenging substrate often requiring harsh reaction conditions (Scheme 3). 28 The reaction was proceeded selectively under mild conditions at room temperature and the corresponding DDSQ 6b bearing four peripheric pyridines was isolated in 84% yield after washing with 5% NH4OH aqueous solution. The structure of this compound was confirmed by multinuclear NMRs and mass spectroscopies. The 29 Si NMR is given as a representative characterization and the four expected singlets (Figure 1d) could be assigned to the silicon atoms from the core (Si in red and green, -79.08 and -78.32 ppm), to the internal D-group silicon atoms (Si in blue, -19.41 ppm) and to the carbosilane-type silicon atoms (Si in pink, 2.80 ppm). Besides, the presence of a singlet at 7.34 ppm ( 1 H NMR) and of a signal at 123.9 ppm ( 13 C NMR) were consistent with the successful formation of the triazole ring. 28 Mass spectroscopy also confirmed the structure. It is worthy to note that the triazole-3-ethynyl moiety possesses interesting properties as ligand for transition metals (Pt, Re for e. g.) and the resulting complexes were shown to present appealing properties in the field of medicine. 29 For example, Pt complexes displayed very high cytotoxicity activity on malignant cells, even higher than cisplatin. Grafting such complexes onto reported DDSQs may lead to nanosized drugs possibly interesting in terms of controlled delivery. Besides, the multivalency, well-known key parameter for biological applications, 24 will be enabled by the DDSQ architecture described in this paper which opens new prospects in this field. More generally, the cycloaddition with other alkynes will offer new opportunities to reach unprecedented multivalent structures having different and new properties as compared to the corresponding monomer, T8, T10 or di-substituted D2T8-analogs.

Conclusion

In summary, a convenient and scalable (gram-scale) route to unprecedented T8D2-DDSQs bearing four chloro-(3b) or four azido (5b) groups was proposed. Both compounds were characterized and proved to undergo successfully nucleophilic substitution for the former (with iodide or azide) and copper-catalyzed azide-alkyne [3+2] cycloaddition for the latter. It is noteworthy that the tetraiodo DDSQ (4b), more reactive than its chloro counterpart, will be of significant interest in the case of poorly reactive nucleophiles. All the transformations occurred under mild conditions and the DDSQs were prepared in very high yields. Beyond the enhanced multivalency as compared to the previously reported di-substituted D2T8 structures, the reported tetra-functional DDSQs are formed as a single isomer and readily isolated in very high yields. Moreover, the tetra-azido DDSQ 5b constitutes a multipurpose nano-building block for the further preparation of new inorganicorganic hybrid materials where the covalent incorporation of a DDSQ moiety brings valuable properties. As mentioned above, the concerned application fields are numerous. To name only a few, the further functionalization of 5b with chromophores, 10 sugars 24 or metal complexes 22 will surely contribute to design new photonic materials for optoelectronic devices, new multivalent bioconjugates for in vivo applications or recyclable catalysts for developing greener processes respectively. Along these lines, studies centered on applications of reported DDSQs are currently underway in our laboratories. EXPERIMENTAL SECTION. All reactions were performed under argon atmosphere using standard Schlenk technique. THF and DMF were dried using mBRAUN purification system.

Toluene was distilled from calcium hydride, stored on 4 Å activated molecular sieves under an argon atmosphere. 2-Butanone was distilled from sodium sulfate, stored on 4 Å activated molecular sieves under an argon atmosphere. (Chloromethyl)dimethylsilane and Karstedt's catalyst (in xylene, 2 % Pt) were purchased from Sigma-Aldrich, sodium azide, sodium ascorbate and 3-ethynylpyridine were purchased from TCI, copper (II) sulfate pentahydrate and sodium iodide were purchased from FUJIFILM Wako Pure Chemical Co. Chemical Reagents and all these reagents were used as received, without further purification. The Fourier transformation nuclear magnetic resonance (NMR) spectra were obtained using a JEOL JNM-ECA 600 ( 1 H at 600.17 MHz, 13 C at 150.91 MHz, 29 Si at 119.24 MHz) NMR instrument. For 1 H NMR, chemical shifts are reported as units (ppm) relative to SiMe4 (TMS) and the residual solvents peaks were used as standards. For 13 C NMR and 29 Si NMR, chemical shifts are reported as units (ppm) relative to SiMe4 (TMS), the residual solvents peaks were used as standards and spectra were obtained with complete proton decoupling. MALDI-TOF mass analysis were carried out with a Shimadzu AXIMA Performance instrument using 2,5-dihydroxybenzoic acid (dithranol) as the matrix. All reagents used were of analytical grade. Elemental analyses were performed by the Center for Material Research by Instrumental Analysis (CIA), Gunma University, Japan. IR spectra were measured with a Shimadzu FTIR-8400S. TGA was carried out under a nitrogen flow (250 mL min - 1 ) with a heating rate 10 ˚C min -1 . All samples were measured with temperatures ranging from 50 to 1000 ˚C, where they remained for 5 min. The weight loss and heating rate were continuously recorded along the experiment.

Synthesis of 3a.

After standard cycles of evacuation and backfilling with dry and pure argon, an oven-dried Schlenk flask equipped with a magnetic stirring bar was charged with 1a (123 mg, 0.1 mmol), distilled toluene (0.6 mL) and (chloromethyl)dimethylsilane 2 (72 L, 0.6 mmol). And then Karstedt's catalyst , 112 L of a pre-prepared [Pt] solution (diluted 100 times from a commercial bottle (2% platinum in xylene) in distilled toluene)] was added at room temperature under argon. After the addition, the reaction mixture was allowed to heat to 40 ˚C and to stir for 16 h at 40 ˚C. The reaction mixture was cooled down to room temperature and then passed through a silica and celite plug which was washed twice with toluene and twice with dichloromethane. The gathered filtrate was evaporated to afford a crude product, which was further purified by recrystallization from chloroform and 2-propanol to give 3a (134 mg, 81%) as a colorless crystal. Analysis data for 3a. MALDI-TOF MS (m/z): 1687.75 ([M+Na] + , calcd 1687.42). Elemental analysis: Calcd for C68H88Cl4O14Si14・2H2O: C, 49.07; H, 5.33. Found: C, 48.03; H, 5.45. 1 H NMR (600.17 MHz, CDCl3): δ = -0.04 (s, 24H), 0.61-0.70 (m, 16H), 2.62 (s, 8H), 7.21-7.29 (m, 16H), 7.37-7.45 (m, 16H), 7.55-7.56 (m, 8H). 13 C{ 1 H} NMR (150.91 MHz, CDCl3): δ = - 5.15, 4.71, 6.93, 29.91, 127.89, 128.01, 130.61, 130.64, 131.11, 132.13, 134.10, 134.18 ppm. 29 Si{ 1 H} NMR (119.24 MHz, CDCl3): δ = 5.36, -19.49, -78.52, -79.60 ppm.

Synthesis of 3b.

After standard cycles of evacuation and backfilling with dry and pure argon, an oven-dried Schlenk flask equipped with a magnetic stirring bar was charged with 1b (643 mg, 0.5 mmol), distilled toluene (3.0 mL) and (chloromethyl)dimethylsilane (365 L, 3.0 mmol). And then Karstedt's catalyst [4.9 x 10 -4 mmol [Pt], 280 L of a pre-prepared [Pt] solution (diluted 50 times from a commercial bottle (2% platinum in xylene) in distilled toluene)] was added at room temperature under argon. After the addition, the reaction mixture was allowed to stir for 16 h at room temperature (25 ˚C). The reaction mixture was passed through a silica and celite plug which was washed twice with toluene and twice with dichloromethane. The gathered filtrate was evaporated to give 3b (860 mg, 99%) as a slightly yellowish solid, without any further purification.

Analysis data for 3b. MALDI-TOF MS (m/z): 1744.03 ([M+Na] + , calcd 1743.53). Elemental analysis: Calcd for C72H96Cl4O14Si14・H2O: C, 49.74; H, 5.68. Found: C, 49.39; H, 5.60. 1 H NMR (600.17 MHz, CDCl3): δ = -0.10 (s, 24H), 0.61-0.64 (m, 8H), 0.82-0.84 (m, 8H), 1.44-1.47 (m, 8H), 2.55 (s, 8H), 7.22-7.28 (m, 16H), 7.37-7.45 (m, 16H), 7.53-7.55 (m, 8H). 13 C{ 1 H} NMR (150.91 MHz, CDCl3): δ = -4. 66, 17.26, 17.97, 20.22, 30.42, 127.90, 128.00, 130.57, 130.61, 131.15, 132.19, 134.07, 134.21 ppm. 29 Si{ 1 H} NMR (119.24 MHz, CDCl3): δ = 3.39, -19.41, -78.27, -79.10 ppm. Note: Hydrosilylation of 1a and 1b exclusively gave rise to the formation of the terminal regioisomer, 3a and 3b, respectively (internally silylated products not observed).

Synthesis of 4a.

After standard cycles of evacuation and backfilling with dry and pure argon, an oven-dried Schlenk flask equipped with a magnetic stirring bar was charged with 3a (167 mg, 0.1 mmol) and distilled 2-butanone (1.3 mL). Sodium iodide (240 mg, 1.6 mmol) was then added into the mixture at room temperature under argon. The reaction mixture was allowed to heat to reflux (80 ˚C) and to stir for 16 h at 80 ˚C. The reaction mixture was cooled down to room temperature and then filtered. The filtrate was evaporated under reduced pressure and then dissolved in chloroform. The solution was washed twice with water and the aqueous layers were extracted twice with chloroform. The organic layers were gathered, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the desired product 4a (201 mg, 99%) as a yellowish solid, without any further purification. Analysis data for 4a. MALDI-TOF MS (m/z): 2053.33 ([M+Na] + , calcd 2053.24). 1 H NMR (600.17 MHz, CDCl3): δ = -0.031 (s, 24H), 0.58-0.60 (m, 8H), 0.67-0.70 (m, 8H), 1.81 (s, 8H), 7.20-7.22 (m, 8H), 7.25-7.28 (m, 8H), 7.36-7.44 (m, 16H), 7.54-7.56 (m, 8H). 13 C{ 1 H} NMR (150.91 MHz,5.90,7.05,127.93,128.02,130.60,130.68,131.13,132.15,134.14,134.24 ppm. 29 Si{ 1 H} NMR (119.24 MHz, CDCl3): δ = 6.98, -19.04, -78.11, -79.19 ppm.

Synthesis of 4b.

After standard cycles of evacuation and backfilling with dry and pure argon, an oven-dried Schlenk flask equipped with a magnetic stirring bar was charged with 3b (172 mg, 0.1 mmol) and distilled 2-butanone (1.3 mL). Sodium iodide (240 mg, 1.6 mmol) was then added into the mixture at room temperature under argon. The reaction mixture was allowed to heated to reflux (80 ˚C) and to stir for 16 h at 80 ˚C. The reaction mixture was cooled down to room temperature and then filtered. The filtrate was evaporated under reduced pressure and then dissolved in chloroform. The solution was washed twice with water and the aqueous layers were extracted twice with chloroform. The organic layers were gathered, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the desired product 4b (207 mg, 99%) as a slightly yellowish gel, without any further purification. Analysis data for 4b. MALDI-TOF MS (m/z): 2109.40 ([M+Na] + , calcd 2109.34). 1 H NMR (600.17 MHz, CDCl3): δ = -0.089 (s, 24H), 0.62-0.65 (m, 8H), 0.80-0.83 (m, 8H), 1.40-1.45 (m, 8H), 1.74 (s, 8H), 7.21-7.28 (m, 16H), 7.36-7.44 (m, 16H), 7.52-7.54 (m, 8H). 13 C{ 1 H} NMR (150.91 MHz, CDCl3): δ = - 12.99, -3.09, 17.40, 19.13, 20.20, 127.95, 128.02, 130.59, 130.63, 131.16, 132.19, 134.09, 134.23 ppm. 29 Si{ 1 H} NMR (119.24 MHz, CDCl3): δ = 4.49, -19.39, -78.32, -79.15 ppm.

Synthesis of 5b.

After standard cycles of evacuation and backfilling with dry and pure argon, an oven-dried Schlenk flask equipped with a magnetic stirring bar was charged with 4b (860 mg, 0.5 mmol), sodium iodide (38 mg, 0.25 mmol) and sodium azide (390 mg, 6.0 mmol). Dry THF (14.4 mL) and dry DMF (36 mL) were then added into the mixture at room temperature under argon with vigorous stirring. The reaction mixture was allowed to heated to 60 ˚C and to stir for 24 h at 60 ˚C. The reaction mixture was cooled down to room temperature, dichloromethane was slowly added and the mixture was stirred for about 10 minutes. And then ice was added into the mixture which was stirred for additional 30 minutes. The organic layer was then washed twice with brine, dried over anhydrous sodium sulfate and evaporated on the rotavapor (with dichloromethane several times to remove traces of DMF) to afford the desired product 5b (804 mg, 92%) as a light yellowish solid, without any further purification. Analysis data for 5b. MALDI-TOF MS (m/z).

1959.82 ([M-4N2+Na] + , calcd 1959.75). 1 H NMR (600.17 MHz, CDCl3): δ = -0.13 (s, 24H), 0.57-0.61 (m, 8H), 0.82-0.84 (m, 8H), 1.41-1.45 (m, 8H), 2.50 (s, 8H), 7.23-7.29 (m, 16H), 7.39-7.46 (m, 16H), 7.52-7.54 (m, 8H). 13 C{ 1 H} NMR (150.91 MHz, CDCl3): δ = -4. 30, 17.24, 18.30, 20.19, 40.95, 127.91, 128.03, 130.64, 130.68, 131.14, 132.14, 134.07, 134.21 ppm. 29 Si{ 1 H} NMR (119.24 MHz, CDCl3): δ = 3. 18, -19.39, -78.25, -79.08 ppm.

Synthesis of 6b.

After standard cycles of evacuation and backfilling with dry and pure argon, an oven-dried Schlenk flask equipped with a magnetic stirring bar was charged with 5b (87 mg, 0.05 mmol), 3-ethynylpyridine (27 mg, 0.27 mmol) and dry THF (1.6 mL). To this solution mixture, 10 L of copper (II) sulfate pentahydrate degassed aqueous solution (62 mg/mL) and sodium ascorbate (3 mg, 0.015 mmol) were added under argon. The solution was allowed to stir at 25 ˚C for 3 days. Additional 10 L of copper sulfate pentahydrate degassed aqueous solution (62 mg/mL) and sodium ascorbate (3 mg, 0.015 mmol) were added under argon into the reaction mixture which was allowed to stir for additional 1 day at 25 ˚C. And then 5% NH4OH aqueous solution was added into the reaction mixture which was extracted 3 times with dichloromethane. The collected organic layer was then washed twice with 5% NH4OH aqueous solution and 3 times with water and dried over anhydrous sodium sulfate. The solvents were evaporated on the rotavapor to give a crude product which was further dried under vacuum at 100 ˚C for 2 hours (to remove the excess of 3ethynylpyridine) to afford the desired product 6b (90 mg, 84%) as a slightly yellowish solid.

Analysis data for 6b. ESI-HRMS: m/z found: 2159.5916 (calculated: 2159.5725). 1 H NMR (600.17 MHz, CDCl3): δ = -0.12 (s, 24H), 0.62-0.66 (m, 8H), 0.77-0.81 (m, 8H), 1.41-1.47 (m, 8H), 3.60 (s, 8H), 7.14-7.17 (m, 8H), 7.21-7.43 (m, 32H), 7.48-7.51 (m, 8H), 8.12 (dt, J = 8.01 Hz, 1.95 Hz, 4H), 8.54 (d, J = 3.21 Hz, 4H), 8.90 (sbr, 4H). 13 C{ 1 H} NMR (150.91 MHz, CDCl3): δ = -4. 15, 17.13, 18.15, 20.02, 40.91, 120.72, 123.92, 127.14, 127.98, 128.10, 130.76, 130.81,

Scheme 2 .

 2 Scheme 2. Synthetic routes to tetrachloro, tetraiodo and tetraazido-DDSQs.

Figure 1 .

 1 Figure 1. 29 Si NMR spectra (in CDCl3) for the DDSQs 3a, 3b, 5b, and 6b. Chemical shifts are in ppm unit.

Figure 2 .

 2 Figure 2. Crystal structure of one fragment of two nonidentical molecules 3a. Black: carbon; blue:

Figure 3 .

 3 Figure 3. Crystal structure of 3b. Black: carbon; blue: silicon; red: oxygen; green: chlorine.

  and SI). Si-O bond lengths (in the ranges of 1.578-1.638 Å; average 1.610 Å) and Si-O-Si bond angles (from 145.4 to 177.5) were found to be similar to those of 3b.

Figure 4 .

 4 Figure 4. Crystal structure of one fragment of two nonidentical molecules 5b. Black: carbon; blue:

 131.01, 131.89, 132.92, 133.96, 134.08, 144.27, 147.03, 149.13 ppm. 29Si{ 1 H} NMR (119.24 MHz, CDCl3): δ = 2.80, - 19.41, -78.32, -79.08 ppm. Crystallography. For crystal data, data collection, and refinement, see SI. CCDC-1971229 for 3a, CCDC-1971230 for 3b, CCDC-1971231 for 4a, CCDC-2021410 for 5b contain the supplementary crystallographic data for this paper.

ASSOCIATED CONTENT

Supporting Information: 1 H, 13 C, 29 Si NMR spectra for compounds 3a, 3b, 4a, 4b, 5b and 6b, IR spectra and thermal properties (TGA) for 3a, 3b, 5b, crystallographic data for 3a, 3b, 4a and 5b. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

Corresponding Authors

Masafumi Unno. Department of Chemistry and Chemical Biology, Graduate School of Science and Technology, Gunma University, Kiryu 376-8515, Gunma, Japan; orcid.org/0000-0003-2158-1644; Email: unno@gunma-u.ac.jp Armelle Ouali. Institut Charles Gerhardt Montpellier ICGM, Univ. Montpellier, CNRS, ENSCM, Montpellier 34090, France; orcid.org/0000-0001-7436-776X. Email: armelle.ouali@enscm.fr.

Authors

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

The authors acknowledge the Gunma University Initiative for Advanced Research (GIAR) for funding the Open International Laboratory and the grant to Y. L, CNRS and New Energy and Industrial Technology Development Organization (NEDO) for funding, and JASSO for M. K. and K. K. travel grants to Montpellier.