Interference-mediated synaptonemal complex formation with embedded crossover designation

Biological systems exhibit complex patterns, at length scales ranging from the molecular to the organismic. Along chromosomes, events often occur stochastically at different positions in different nuclei but nonetheless tend to be relatively evenly spaced. Examples include replication origin firings, formation of chromatin loops along chromosome axes and, during meiosis, designation of crossover recombination sites ("crossover interference"). We present evidence, in the fungus Sordaria macrospora, that crossover interference is part of a broader patterning program that includes synaptonemal complex (SC) nucleation. This program yields relatively evenly-spaced SC nucleation sites; among these, a subset is also crossover sites that show a classical interference distribution. This pattern ensures that SC forms regularly along the entire lengths of the chromosomes as required for homolog pairing maintenance and interlock sensing while concomitantly embedding crossover interactions within the SC structure as required for both DNA recombination and structural events of chiasma-formation. This pattern can be explained by a threshold-based interference process. This model can be generalized to give diverse types of related and/or partially overlapping patterns, in two or more dimensions, for any type of object.Other Research Uni

Identification of ASYNAPTIC4, a Component of the Meiotic Chromosome Axis

International audienceDuring the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes. Most meiotic recombination intermediates that give rise to interhomolog crossovers are embedded within a hallmark chromosomal structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known structural components of the budding yeast SC, the Zip1 protein is unique for its independent role in promoting crossover recombination; Zip1 is specifically required for the large subset of crossovers that also rely on the meiosis-specific MutSgamma complex. Here we report that adjacent regions within Zip1\u27s N terminus encompass its crossover and synapsis functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSgamma crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSgamma crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSgamma crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3\u27s localization at Zip1 polycomplex and supports some MutSgamma crossing over but prevents normal SC assembly and Ecm11 SUMOylation. Our results highlight distinct N terminal regions that are differentially critical for Zip1\u27s roles in crossing over and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors within close proximity of one another

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

A High Throughput Genetic Screen Identifies New Early Meiotic Recombination Functions in Arabidopsis thaliana

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes

Etude de l'initiation de la recombinaison méiotique chez Arabidopsis thaliana

La méoise est une étape obligatoire pour les organismes se reproduisant de façon sexuée. Elle comprend une phase de réplication de l ADN suivie de deux étapes de ségrégation chromosomique. Pour que ce processus se déroule correctement, un certain nombre d événements doivent avoir lieu, parmi lesquels la recombinaison homologue joue un rôle essentiel. La recombinaison homologue méiotique débute par la formation de cassures double brin (CDB) de l ADN initiées par la protéine Spo11. Chez les plantes, peu de choses sont connues sur cette étape de la recombinaison puisque, mis à part AtPO11-1 et AtPO11-2, aucun autre gène impliqué dans la formation des CDB n a été identifié. Une partie de cette thèse a consisté à rechercher de nouveaux marqueurs de CDB. Dans ce contexte, le comportement de la protéine AtMRE11 et de l histone H2AX phosphorylée (yH2AX) ont été entrepris. Ces études ont montré que ces protéines sont localisées sur les chromosomes au cours de la prophase méiotique. Cependant, elles ne peuvent être utilisées comme marqueurs de CDB puisque leur localisation n est pas dépendante de l initiation de la recombinaison. Parallèlement, l objectif principal de la thèse a été d identifier et de caractériser des protéines intervenant dans les étapes précoces de la recombinaison homologue en utilisant comme modèle Arabidopsis thaliana. Cette étude s est basée sur la recherche de mutants phénocopiant la mutation Atspo11-1 au sein d une collection de mutants issus d une mutagénèse à saturation (une mutation toutes les 250 pb). Les résultats de ce travail ont montré qu au moins trois nouvelles protéines, AtPRD1, AtPRD2 et AtPRD3 (pour Arabidopsis thaliana Putative initiation Recombination Defects 1-3) sont requises pour la formation des CDB. De plus, AtPRD1, AtPDR2 et AtSPO11-1 interagissent en double hybride, ce qui suggère qu il existe un complexe d initiation de la recombinaison chez Arabidopsis. Enfin, le crible mené nous a permis d approfondir la fonction de la protéine SDS (Solo Dancers, Azumi et al., 2002) en montrant qu elle intervenait dans le choix du partenaire pendant la réparation des CDBs méiotiques (biais interhomologue) au même titre que ASY1 et AtDMC1.Meiosis is a crucial step for organisms that reproduce sexually. It is a characterized by a single pound of DNA replication followed by two steps of chromosomal segregation. For this, specific events of meiosis need to take place. Among them, homologous recombination plays an essential role in numerous species, including Arabidopsis thaliana. Homologous recombination starts by the formation of DNA double strand breaks (DSB) generated by Spo11. In plants, AtSPO11-1 and AtSPO11-2, are the only genes known to be necessary for DSB formation. The main objective of my PhD was to isolate and characterise genes involved in DSB formation using Arabidopsis thaliana as a model. Simultaneously, analysis of new DSB markers, such as yH2AX and AtMRE11, was undertaken. In this work, studies of yH2AX and AtMRE11 immunolabelling showed that these proteins are localized on chromosomes during meiotic prophase l. However, they cannot be used as DSB markers since their localization is independent of meiotic recombination initiation. The identification of new proteins involved in DSB formation was undertaken by looking for mutants showing a typical Atspo11-1 phenotype among a saturated mutant collection of Arabidopsis (one mutation every 250 bp). Our results show that at least three new proteins, AtPRD1, AtPRD2 and AtPRD3 (for Arabidopsis thaliana Putative initiation Recombination Defects 1-3) are involved in DSB formation. Furthermore, AtPRD1, AtPRD2 and AtSPO11-1 interact in a yeast two hybrid assay, suggesting that a DSB formation complex exists in Arabidopsis. Finally, our screen provided new insights concerning the function of the SDS protein (Solo Dancers Azumi et al., 2002) showing that it is required for partner choice during meiotic DSB repair (interhomolog bias) as well as ASY1 and AtDMC1.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

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

AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

International audienceThe initiation of meiotic recombination by the formation of DNA double-strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11-1 interact in a yeast two-hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11-1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue