A meiotic XPF-ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2–Zip4–Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF–ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals

Interaction entre les crossing-overs méiotiques et l’architecture du chromosome : rôle du complexe spécifique de la méiose Zip2-Zip4-Spo16

Meiosis is a highly conserved mechanism among organisms with sexual development. This process consists in producing four haploid gametes from one diploid cell by executing two successive rounds of cell division. During the first meiotic division, reciprocal exchanges of parental DNA strands, also known as crossing-overs (COs), ensure the faithful segregation of homologous chromosomes. COs arise from a specific type of DNA repair, homologous recombination. This pathway is initiated by the simultaneous induction of hundreds of double strand breaks (DSBs) in the genome. In budding yeast, the major CO pathway is promoted by a family of eight conserved proteins, named ZMMs (acronym for Zip1/2/3/4-Msh4/5-Mer3-Spo16), involved in recognizing and stabilizing DNA intermediates formed during homologous recombination. We showed that the Zip4 protein forms a stable tripartite complex with two other ZMM proteins, Zip2 and Spo16. Our data suggests that the Zip2-Zip4-Spo16 (ZZS) complex binds recombination intermediates through its XPF-ERCC1-like domain and drives them towards a CO fate. The homologs of Zip2 and Zip4 in mammals, SHOC1 and TEX11 respectively, have been described, but no Spo16 homolog has been found so far. We could identify the homolog of Spo16 in mammals by an in silico screen, MmSPO16. In addition, I could co-purify MmSPO16 with the XPF domain of SHOC1, thus revealing the potential conservation of the entire ZZS complex in mammals. ZMM-dependent COs are formed within the context of a meiosis-specific structure, named synaptonemal complex (SC). The SC is a proteinaceous structure composed of two axial elements physically maintained together at a precise distance of 100 nm by a central region. The central region encompasses a central element, composed of the two proteins Ecm11 and Gmc2, and the transverse filaments composed of Zip1. The transverse filaments from opposing axial elements overlap and bind head-to-head in the central element. However, despite evidence of a close relationship between SC assembly and CO formation, nothing is known about a direct link that could coordinate these two events spatially and temporally. During my PhD, I found a new interaction between the SC protein Ecm11 and the ZMM protein Zip4. This newly discovered interaction is necessary for Ecm11 association and polymerization on chromosomes, the SC assembly and the homolog disjunction in meiosis I. Our results suggest a direct connection that ensures SC assembly from CO sites through the Zip4-Ecm11 interaction. This way, ensuring SC polymerization from emerging CO sites could be a way of fine-tuning CO distribution, by participating to CO interference and/or by regulating nearby DSB formation. Moreover, I could identify an interaction between the mammalian ortholog of Zip4, TEX11, and one of the five members composing the SC central element, TEX12, raising the possibility that this mechanism synchronizing CO formation and SC polymerization could be conserved.La méiose est une étape essentielle de la reproduction chez tous les organismes sexués. En effet, celle-ci permet l’obtention de quatre gamètes haploïdes à partir d’une seule cellule diploïde grâce à la réalisation deux divisions successives suivant une seule étape de réplication. Un des éléments essentiels permettant une bonne ségrégation en première division méiotique est la création d’un échange physique entre les chromosomes homologues parentaux. Ce lien physique, plus communément appelé crossing-over (CO), est produit par un mécanisme de recombinaison entre les chromosomes homologues au cours de la prophase I méiotique. La recombinaison homologue est initiée par la formation simultanée de nombreuses cassures double-brin au sein du génome. Chez la levure de boulanger, la formation des COs est dépendante de la famille protéique ZMM (un acronyme pour Zip1/2/3/4-Msh4/5-Mer3-Spo16) composée de huit protéines hautement conservées, et impliquées dans la reconnaissance et la stabilisation des intermédiaires d’ADN formés au cours de la recombinaison homologue. Nous avons montré que la protéine Zip4 forme un complexe stable avec deux autres protéines ZMM, Zip2 et Spo16. Le complexe Zip2-Zip4-Spo16 (ZZS), de type XPF-ERCC1, serait capable de reconnaitre et de stabiliser les intermédiaires de recombinaison afin de promouvoir leur réparation en tant que CO. Chez les mammifères, Zip2 et Zip4 possèdent des homologues décrits, SHOC1 et TEX11 respectivement, mais aucun homologue n’a été découvert pour Spo16. Nous avons réalisé une analyse in silico et pu déterminer un homologue de Spo16 chez les mammifères, MmSPO16. Par la suite, j’ai pu co-purifier MmSPO16 avec le domaine XPF de SHOC1, ce qui suggère la conservation du complexe ZZS chez les mammifères. De plus, le processus de formation des COs est corrélé́ à la mise en place d’un complexe protéique formé entre les deux chromosomes homologues, appelé complexe synaptonémal (CS). Le CS est composé de deux éléments axiaux, accolés entre eux à une distance précise de 100 nm par la région centrale. La région centrale comprend un élément central, composé de l’hétérodimère Ecm11-Gmc2, et d’un élément transversal formé par la protéine Zip1. Les éléments transversaux partant des axes opposés se lient tête-bêche au niveau de l’élément central. Malgré des liens fonctionnels évidents entre la formation des COs et l’assemblage du CS entre les chromosomes homologues, aucun lien physique direct n’a été établi à ce jour. Au cours de mon doctorat, j’ai pu démontrer l’existence d’une interaction physique entre la protéine du CS Ecm11 et la protéine ZMM Zip4. Cette interaction est nécessaire pour la localisation et la polymérisation d’Ecm11 sur les chromosomes, l’assemblage correct du CS et la ségrégation des chromosomes homologues en première division méiotique

Interaction entre les crossing-overs méiotiques et l’architecture du chromosome : rôle du complexe spécifique de la méiose Zip2-Zip4-Spo16

Meiosis is a highly conserved mechanism among organisms with sexual development. This process consists in producing four haploid gametes from one diploid cell by executing two successive rounds of cell division. During the first meiotic division, reciprocal exchanges of parental DNA strands, also known as crossing-overs (COs), ensure the faithful segregation of homologous chromosomes. COs arise from a specific type of DNA repair, homologous recombination. This pathway is initiated by the simultaneous induction of hundreds of double strand breaks (DSBs) in the genome. In budding yeast, the major CO pathway is promoted by a family of eight conserved proteins, named ZMMs (acronym for Zip1/2/3/4-Msh4/5-Mer3-Spo16), involved in recognizing and stabilizing DNA intermediates formed during homologous recombination. We showed that the Zip4 protein forms a stable tripartite complex with two other ZMM proteins, Zip2 and Spo16. Our data suggests that the Zip2-Zip4-Spo16 (ZZS) complex binds recombination intermediates through its XPF-ERCC1-like domain and drives them towards a CO fate. The homologs of Zip2 and Zip4 in mammals, SHOC1 and TEX11 respectively, have been described, but no Spo16 homolog has been found so far. We could identify the homolog of Spo16 in mammals by an in silico screen, MmSPO16. In addition, I could co-purify MmSPO16 with the XPF domain of SHOC1, thus revealing the potential conservation of the entire ZZS complex in mammals. ZMM-dependent COs are formed within the context of a meiosis-specific structure, named synaptonemal complex (SC). The SC is a proteinaceous structure composed of two axial elements physically maintained together at a precise distance of 100 nm by a central region. The central region encompasses a central element, composed of the two proteins Ecm11 and Gmc2, and the transverse filaments composed of Zip1. The transverse filaments from opposing axial elements overlap and bind head-to-head in the central element. However, despite evidence of a close relationship between SC assembly and CO formation, nothing is known about a direct link that could coordinate these two events spatially and temporally. During my PhD, I found a new interaction between the SC protein Ecm11 and the ZMM protein Zip4. This newly discovered interaction is necessary for Ecm11 association and polymerization on chromosomes, the SC assembly and the homolog disjunction in meiosis I. Our results suggest a direct connection that ensures SC assembly from CO sites through the Zip4-Ecm11 interaction. This way, ensuring SC polymerization from emerging CO sites could be a way of fine-tuning CO distribution, by participating to CO interference and/or by regulating nearby DSB formation. Moreover, I could identify an interaction between the mammalian ortholog of Zip4, TEX11, and one of the five members composing the SC central element, TEX12, raising the possibility that this mechanism synchronizing CO formation and SC polymerization could be conserved.La méiose est une étape essentielle de la reproduction chez tous les organismes sexués. En effet, celle-ci permet l’obtention de quatre gamètes haploïdes à partir d’une seule cellule diploïde grâce à la réalisation deux divisions successives suivant une seule étape de réplication. Un des éléments essentiels permettant une bonne ségrégation en première division méiotique est la création d’un échange physique entre les chromosomes homologues parentaux. Ce lien physique, plus communément appelé crossing-over (CO), est produit par un mécanisme de recombinaison entre les chromosomes homologues au cours de la prophase I méiotique. La recombinaison homologue est initiée par la formation simultanée de nombreuses cassures double-brin au sein du génome. Chez la levure de boulanger, la formation des COs est dépendante de la famille protéique ZMM (un acronyme pour Zip1/2/3/4-Msh4/5-Mer3-Spo16) composée de huit protéines hautement conservées, et impliquées dans la reconnaissance et la stabilisation des intermédiaires d’ADN formés au cours de la recombinaison homologue. Nous avons montré que la protéine Zip4 forme un complexe stable avec deux autres protéines ZMM, Zip2 et Spo16. Le complexe Zip2-Zip4-Spo16 (ZZS), de type XPF-ERCC1, serait capable de reconnaitre et de stabiliser les intermédiaires de recombinaison afin de promouvoir leur réparation en tant que CO. Chez les mammifères, Zip2 et Zip4 possèdent des homologues décrits, SHOC1 et TEX11 respectivement, mais aucun homologue n’a été découvert pour Spo16. Nous avons réalisé une analyse in silico et pu déterminer un homologue de Spo16 chez les mammifères, MmSPO16. Par la suite, j’ai pu co-purifier MmSPO16 avec le domaine XPF de SHOC1, ce qui suggère la conservation du complexe ZZS chez les mammifères. De plus, le processus de formation des COs est corrélé́ à la mise en place d’un complexe protéique formé entre les deux chromosomes homologues, appelé complexe synaptonémal (CS). Le CS est composé de deux éléments axiaux, accolés entre eux à une distance précise de 100 nm par la région centrale. La région centrale comprend un élément central, composé de l’hétérodimère Ecm11-Gmc2, et d’un élément transversal formé par la protéine Zip1. Les éléments transversaux partant des axes opposés se lient tête-bêche au niveau de l’élément central. Malgré des liens fonctionnels évidents entre la formation des COs et l’assemblage du CS entre les chromosomes homologues, aucun lien physique direct n’a été établi à ce jour. Au cours de mon doctorat, j’ai pu démontrer l’existence d’une interaction physique entre la protéine du CS Ecm11 et la protéine ZMM Zip4. Cette interaction est nécessaire pour la localisation et la polymérisation d’Ecm11 sur les chromosomes, l’assemblage correct du CS et la ségrégation des chromosomes homologues en première division méiotique

Crossing and zipping: molecular duties of the ZMM proteins in meiosis

International audienceAccurate segregation of homologous chromosomes during meiosis depends on the ability of meiotic cells to promote reciprocal exchanges between parental DNA strands, known as crossovers (COs). For most organisms, including budding yeast and other fungi, mammals, nematodes, and plants, the major CO pathway depends on ZMM proteins, a set of molecular actors specifically devoted to recognize and stabilize CO-specific DNA intermediates that are formed during homologous recombination. The progressive implementation of ZMM-dependent COs takes place within the context of the synaptonemal complex (SC), a proteinaceous structure that polymerizes between homologs and participates in close homolog juxtaposition during prophase I of meiosis. While SC polymerization starts from ZMM-bound sites and ZMM proteins are required for SC polymerization in budding yeast and the fungus Sordaria, other organisms differ in their requirement for ZMM in SC elongation. This review provides an overview of ZMM functions and discusses their collaborative tasks for CO formation and SC assembly, based on recent findings and on a comparison of different model organisms

The Zip4 protein directly couples meiotic crossover formation to synaptonemal complex assembly

International audienceMeiotic recombination is triggered by programmed double-strand breaks (DSBs), a subset of these being repaired as crossovers, promoted by eight evolutionarily conserved proteins, named ZMM. Crossover formation is functionally linked to synaptonemal complex (SC) assembly between homologous chromosomes, but the underlying mechanism is unknown. Here we show that Ecm11, a SC central element protein, localizes on both DSB sites and sites that attach chromatin loops to the chromosome axis, which are the starting points of SC formation, in a way that strictly requires the ZMM protein Zip4. Furthermore, Zip4 directly interacts with Ecm11, and point mutants that specifically abolish this interaction lose Ecm11 binding to chromosomes and exhibit defective SC assembly. This can be partially rescued by artificially tethering interaction-defective Ecm11 to Zip4. Mechanistically, this direct connection ensuring SC assembly from CO sites could be a way for the meiotic cell to shut down further DSB formation once enough recombination sites have been selected for crossovers, thereby preventing excess crossovers. Finally, the mammalian ortholog of Zip4, TEX11, also interacts with the SC central element TEX12, suggesting a general mechanism

A meiotic XPF–ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2–Zip4–Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF–ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals.ISSN:0890-9369ISSN:1549-547