Exo1 recruits Cdc5 polo kinase to MutLγ to ensure efficient meiotic crossover formation Aurore Sancheza,b,c, Céline Adama,b, Felix Rauhd, Yann Duroca,b, Lepakshi Ranjhac, Bérangère Lombarde, Xiaojing Muf,g, Mélody Wintreberta,b, Damarys Loewe, Alba Guarnéh, Stefano Gnana,b, Chun-Long Chena,b, Scott Keeneyf,g,i, Petr Cejkac, Raphaël Guéroisj, Franz Kleind, Jean-Baptiste Charbonnierj, and Valérie Bordea,b,1 aInstitut Curie, Paris Sciences et Lettres Research University, CNRS, UMR3244, 75005 Paris, France; bParis Sorbonne Université, UMR3244, 75005 Paris, France; cInstitute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera Italiana, 6501 Bellinzona, Switzerland; dMax F. Perutz Laboratories, University of Vienna, 1010 Wien, Austria; eInstitut Curie, Paris Sciences et Lettres Research University, 75006 Paris, France; fMolecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065; gWeill Graduate School of Medical Sciences, Cornell University, New York, NY 10065; hDepartment of Biochemistry, Centre de Recherche en Biologie Structurale, McGill University, Montreal, QC H3A 0G4, Canada; iMemorial Sloan Kettering Cancer Center, Howard Hughes Medical Institute, New York, NY 10065; and jInstitute for Integrative Biology of the Cell (I2BC), Commissariat à l’Energie Atomique, CNRS, Université Paris-Sud, Université Paris-Saclay, 91190 Gif-sur-Yvette, France Edited by Anne M. Villeneuve, Stanford University, Stanford, CA, and approved October 14, 2020 (received for review July 7, 2020) Crossovers generated during the repair of programmed meiotic recruits a MutL-related complex to initiate repair together with double-strand breaks must be tightly regulated to promote accu- proliferating cell nuclear antigen and Exo1 (reviewed in ref. 11). rate homolog segregation without deleterious outcomes, such as MutLα represents the major mismatch repair activity, which in- aneuploidy. The Mlh1–Mlh3 (MutLγ) endonuclease complex is crit- volves its endonuclease activity (12, 13). The related MutLγ is ical for crossover resolution, which involves mechanistically un- instead essential to meiotic crossover, a process that remains clear interplay between MutLγ and Exo1 and polo kinase Cdc5. relatively ill-defined, although MutLγ endonuclease activity is Using budding yeast to gain temporal and genetic traction on required for crossover (9, 14–16). Despite its importance and crossover regulation, we find that MutLγ constitutively interacts cross-species relevance, the mechanism of dHJ resolution and with Exo1. Upon commitment to crossover repair, MutLγ–Exo1 as- crossover formation by MutLγ remains unknown. MutLγ alone sociate with recombination intermediates, followed by direct Cdc5 does not resolve HJs in vitro (15–17), and may need additional recruitment that triggers MutLγ crossover activity. We propose partners, meiosis-specific posttranslational modifications, and a GENETICS that Exo1 serves as a central coordinator in this molecular inter- specific DNA substrate to promote specific nuclease activity and play, providing a defined order of interaction that prevents dele- hence crossover formation. γ terious, premature activation of crossovers. MutL associates at a Based on recent in vitro experiments and genome-wide analysis lower frequency near centromeres, indicating that spatial regula- of meiotic recombination, it seems that rather than acting as a tion across chromosomal regions reduces risky crossover events. canonical resolvase, MutLγ may nick the DNA, which could result Our data elucidate the temporal and spatial control surrounding a in dHJ resolution and crossover formation if two closely spaced constitutive, potentially harmful, nuclease. We also reveal a criti- nicks on opposite strands are made (17, 18). In vitro analyses with cal, noncatalytic role for Exo1, through noncanonical interaction recombinant proteins also suggest that MutLγ mayneedtoex- with polo kinase. These mechanisms regulating meiotic crossovers tensively polymerize along DNA in order to activate its DNA may be conserved across species. cleavage activity (17). recombination | meiosis | crossovers | polo kinase | MutL Significance eiotic recombination provides crossovers that allow homol- ogous chromosomes to be transiently physically attached Meiotic crossovers are essential for the production of gametes M γ – together and then properly segregated (1). A lack or altered dis- with balanced chromosome content. MutL (Mlh1 Mlh3) endo- tribution of meiotic crossovers is a major source of aneuploidies, nuclease, a mismatch repair heterodimer, also functions during meiosis to generate crossovers. Its activity requires Exo1 as well as leading to sterility or disorders, such as Down syndrome, providing γ – strong impetus for understanding how crossovers are controlled the MutS heterodimer (Msh4 Msh5). Crossovers also require the during meiosis. Meiotic recombination is triggered by the forma- polo kinase Cdc5 in a way that is poorly understood. We show that Exo1 directly interacts with Cdc5, and that this promotes the tion of programmed DNA double-strand breaks (DSBs), catalyzed γ by Spo11 together with several conserved protein partners (2). activation of MutL for crossovers. Since Cdc5 also controls other key meiotic processes, including kinetochores mono-orientation Following Spo11 removal, 5′ DSB ends are resected and the 3′ and disassembly of the synaptonemal complex, this direct inter- ends invade a homologous template, leading to formation of action with the crossover proteins may be a way to coordinate D-loop intermediates. After DNA synthesis, many of these inter- meiotic chromosome segregation events to avoid untimely acti- mediates are dismantled by helicases and repaired without a vation of the MutLγ endonuclease once recruited to future crossover (3). However, a subset is stabilized, and upon capture of crossover sites. the second DSB end, mature into a double Holliday junction (dHJ), which is almost exclusively resolved as a crossover (4, 5). Author contributions: A.S., D.L., S.K., P.C., R.G., F.K., J.-B.C., and V.B. designed research; Essential to this crossover pathway are a group of proteins, called A.S., C.A., F.R., Y.D., L.R., B.L., X.M., M.W., A.G., and V.B. performed research; S.K. ZMMs, which collectively stabilize and protect recombination in- contributed new reagents/analytic tools; A.S., C.A., F.R., Y.D., L.R., B.L., M.W., D.L., termediates from helicases (3, 6–8). The mismatch repair MutLγ S.G., C.-L.C., F.K., and V.B. analyzed data; and A.S. and V.B. wrote the paper. (Mlh1–Mlh3) heterodimer is proposed to subsequently act on The authors declare no competing interest. these ZMM-stabilized dHJs to resolve them into crossovers (9). This article is a PNAS Direct Submission. Accordingly, MutLγ foci reflect the number and distribution of Published under the PNAS license. crossovers on both mammalian and plant meiotic chromosomes 1To whom correspondence may be addressed. Email: [email protected]. (reviewed in ref. 10). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ In a distinct process, eukaryotic mismatch repair, a MutS- doi:10.1073/pnas.2013012117/-/DCSupplemental. related complex (MutSα or MutSβ) recognizes a mismatch, and www.pnas.org/cgi/doi/10.1073/pnas.2013012117 PNAS Latest Articles | 1of12 Downloaded by guest on September 29, 2021 MutLγ partners with the ZMM MutSγ heterodimer (Msh4– with the three tested DSB hotspots during meiosis, reaching a Msh5), proposed to first stabilize recombination intermediates maximum at the expected time of crossover formation (4 to 5 h) and then to work together with MutLγ for crossover resolution (Fig. 2A and SI Appendix,Fig.S2A) (5), with kinetics very similar (reviewed in refs. 8 and 11). In addition, the exonuclease Exo1 to the ZMM proteins (33, 34). Mlh3 also weakly associated with interacts in vitro with Mlh1. Previous work showed that Exo1’s the chromosome axis, where DSB sites transiently relocate during ability to interact with Mlh1 is important for MutLγ function in recombination (35, 36) (Fig. 2A). Mlh3 association with DSB crossover formation (19). This leaves the mechanism and role of hotspots required DSB formation, since it was not observed in a Exo1 in meiotic crossovers unclear, although broad genetic spo11Δ mutant (Fig. 2A and SI Appendix,Fig.S2B), but was in- analysis suggests a noncatalytic role given that Exo1-null yeast dependent of its nuclease activity (mlh3-D523N mutant) (Fig. 2A show reduced crossovers and reduced viable gametes, but cata- and SI Appendix, Fig. S2C). Mlh3 binding also required the ZMM lytic mutants of Exo1 do not (9, 20). Similarly, Exo1-null mice protein, Mer3 (Fig. 2A and SI Appendix,Fig.S2D). In msh4Δ are sterile, but not Exo1 catalytic mutants, highlighting the mutants, Mlh3 recruitment was strongly reduced (Fig. 2A and SI conservation of this function (21, 22). How Exo1 is required for Appendix,Fig.S2E), consistent with the reduced number of Mlh3 activating MutLγ is not known. Finally, the polo-like kinase Cdc5 foci in msh4Δ (Fig. 1 G and H). In contrast, Mlh3 bound at (homolog of human PLK1) is required for meiotic crossovers (23). hotspots at nearly wild-type levels in exo1Δ mutants (Fig. 2A Cdc5 expression is induced by the Ndt80 transcription factor at the and SI Appendix,Fig.S2F). Finally, to understand how and pachytene stage of meiotic prophase, once homolog synapsis and when it functions, we assessed the role of Cdc5 polo kinase, – recombination intermediates