Manifestation visible des cross-overs génétiques, les chiasmata lient les paires de chromosomes homologues afin de les orienter correctement sur le fuseau méiotique en Métaphase et Anaphase I. Ils résultent d'un processus complexe et étroitement régulé impliquant l'induction de cassures double-brins et de leur réparation par l'invasion d'un duplex d'ADN homologue faisant office de modèle. La recombinaison est ainsi essentielle pour le synapsis et la ségrégation correcte des chromosomes méiotiques à la première division méiotique, et pour la génération de la variabilité génétique. Bien que les processus permettant à un chromosome de s'apparier seulement à son homologue ne soient pas complètement élucidés, l'appariement des chromosomes homologues est étroitement lié à la recombinaison catalysée par les enzymes d'échange de brins d'ADN RAD51 et DMC1. Ces deux protéines ont des capacités très similaires in vitro, mais sont fonctionnellement distinctes in vivo.La première partie de ma thèse montre l'impact de l'élimination de l'activité d'échange de brins de RAD51 dans la méiose d'Arabidopsis, tout en conservant sa fonction de facteur accessoire pour l'action de DMC1. La recombinaison peut donner lieu à des cross-over (CO) et non-cross-over (NCO) et la recombinase spécifique de la méiose DMC1 a été jugée particulièrement importante dans la production de CO interhomologue. Des résultats récents suggèrent fortement toutefois que DMC1 est la seule recombinase active dans la méiose et doit donc être responsable des résultats de CO et NCO. Etant donné qu'environ 95% de la recombinaison méiotique homologue dans Arabidopsis n'entraîne pas de cross-overs interhomologues, Arabidopsis est un modèle particulièrement sensible pour tester l'importance relative des deux protéines - même des effets mineurs sur la population d'événements non-cross-over devraient produire des effets détectables sur les cross-overs. DMC1 catalyse la réparation de toutes les cassures d'ADN méiotiques en présence d'une protéine RAD51 catalytiquement inactive (fusion RAD51-GFP), et les résultats de mon travail montrent que cela n'a pas d'effet détectable sur les taux relatifs de recombinaison de CO et de NCO : à la fois localement, à l'échelle du chromosome et du génome. Et non plus sur la progression de la division méiotique. Ce travail a abouti à une publication dans le journal PLoS One (Singh G, Da Ines O, Gallego ME & White CI (2017) Analyse de l'impact de l'absence d'activité d'échange de brins de RAD51 dans la méiose d'Arabidopsis PLoS ONE 12: e0183006- 16).Des publications antérieures montrent une synapsis homologue partielle et incomplète en l'absence de rad51 et xrcc3 dans la méiose d'Arabidopsis. Cela s'accompagne de la présence de nombreuses fibres courtes ZYP1 dans ces noyaux, ce qui pourrait indiquer de faibles longueurs de complèxe synaptonémale (SC). Ce synapsis partielle dépend à la fois de SPO11 et de DMC1 et implique des péricentromères, montrant que DMC1 est capable (au moins partiellement) d'entraîner le synapsis dans les péricentromères en l'absence de RAD51. Afin de mieux caractériser ceci et pour tester l'hypothèse que les fibres ZYP1 courtes montrent la présence d'une initiation de SC à ces sites, j'ai méné des expériences d'immunofluorescence et d'imagerie SIM. Utilisant un coloration DAPI et les antiséra ASY1, ZYP1 et CENH3, j'ai conduite des analyses cytogénétiques de le synapsis dans les mutants rad51, xrcc3 et des plantes sauvages. Ces travaux faisaient l'objet de la deuxième partie de mes travaux de thèse. Dans les plantes mutantes, j'observe effectivement des fibres courtes ZYP1 comprenant des centromères, mais elles ne sont pas la règle, ce qui signifie que le synapsis ne commence pas nécessairement à des centromères ou des péricentromères. (...) / The visible manifestation of genetic crossing-over, chiasmata link homologous chromosome pairs to permit them to properly orient on the meiotic Anaphase I spindle. They are the result of an intricate and tightly regulated process involving induction of DNA double- strand breaks and their repair through invasion of a homologous template DNA duplex. Recombination is thus essential for the synapsis and accurate segregation of meiotic chromosomes at the first meiotic division, and in doing so, generates genetic variation. Although the processes permitting a chromosome to pair only with its homologue are not fully understood, successful pairing of homologous chromosomes is tightly linked to recombination catalysed by the DNA strand exchange enzymes RAD51 and DMC1. Both proteins share very similar capabilities in vitro, but are functionally distinct in vivo. The first part of my thesis shows the impact of eliminating the strand exchange activity of RAD51 in Arabidopsis meiosis, while retaining its function as an accessory factor for the action of DMC1. Recombination can give rise to both crossover (CO) and non-crossover (NCO) outcomes and the meiosis-specific recombinase DMC1 has been thought to be of particular importance in the production of inter-homolog CO. Recent results however suggest strongly that that DMC1 is the only active recombinase in wild-type meiosis and thus must be responsible for both CO and NCO outcomes. Approximately 95% of meiotic homologous recombination in Arabidopsis does not result in inter-homologue crossovers and Arabidopsis is thus a particularly sensitive model for testing the relative importance of the two proteins - even minor effects on the non-crossover event population should produce detectable effects on crossing-over. DMC1 catalyses repair of all meiotic DNA breaks in the presence of the catalytically inactive RAD51 (RAD51-GFP fusion) and the results of my work show that this has no detectable effect on the relative rates of CO and NCO recombination, both locally and chromosome- and genome-wide, nor on the progression of the meiotic division. This work has resulted in a publication in the journal PLoS One (Singh G, Da Ines O, Gallego ME & White CI (2017) Analysis of the impact of the absence of RAD51 strand exchange activity in Arabidopsis meiosis. PLoS ONE 12: e0183006–16).Previous publications show partial, incomplete homolog synapsis in the absence of rad51 and xrcc3 in Arabidopsis meiosis. This is accompanied by the presence of many short ZYP1 fibres in these nuclei, possibly indicating short stretches of Synaptonemal Complex (SC). The partial synapsis is both SPO11- and DMC1-dependent and involves peri-centromeres, showing that DMC1 is able to (at least partially) drive synapsis in peri-centromeres in the absence of RAD51. In an effort to better characterize this and to test the hypothesis that the short ZYP1 fibres show the presence of initiation of SC at these sites, immunofluorescence and SIM imaging with DAPI staining and ASY1, ZYP1 and CENH3 antisera were carried out for cytogenetic analyses of synapsis in rad51 and xrcc3 mutants and the WT in the second part of my thesis work. Although I do observe short ZYP1 fibres including centromeres in the mutants, these are not the rule, so synapsis does not necessarily begin at centromeres or peri-centromeres. The superresolution imaging does confirm the presence of stretches of 4-chromatid fibres in xrcc3 plants and this approach will be extended in future work of the group to probe the nature of the RAD51-independent partial meiotic chromosome synapsis.Finally, I have designed and built CRISPR/CAS9 constructs with the aim of creating meiotic DSB hotspots at specific genomic loci. Taking advantage of single nucleotide polymorphism data, these constructs were designed to specifically cleave sites in the Arabidopsis Col-0 ecotype, and not in Ler-0 plants. (...)

Meiosis is necessary for sexual reproduction in eukaryotes. Genetic recombination between non-sister homologous chromosomes is needed in most organisms for successful completion of the first meiotic division. Proteins that function during meiotic recombination have been studied extensively in model organisms. However, less is known about the evolution of these proteins, especially among protists. We searched the genomes of diverse eukaryotes, representing all currently recognized supergroups, for 26 genes encoding proteins important for different stages of interhomolog recombination. We also performed phylogenetic analyses to determine the evolutionary relationships of gene homologs. At least 23 of the genes tested (nine that are known to function only during meiosis in model organisms) are likely to have been present in the Last Eukaryotic Common Ancestor (LECA). These genes encode products that function during: i) synaptonemal complex formation; ii) interhomolog DNA strand exchange; iii) Holliday junction resolution; and iv) sister-chromatid cohesion. These data strongly suggest that the LECA was capable of these distinct and important functions during meiosis. We also determined that several genes whose products function during both mitosis and meiosis are paralogs of genes whose products are known to function only during meiosis. Therefore, these meiotic genes likely arose by duplication events that occurred prior to the LECA. The Rad51 protein catalyzes DNA strand exchange during both mitosis and meiosis, while Dmc1 catalyzes interhomolog DNA strand exchange only during meiosis. To study the evolution of these important proteins, we performed degenerate PCR and extensive nucleotide and protein sequence database searches to obtain data from representatives of all available eukaryotic supergroups. We also performed phylogenetic analyses on the Rad51 and Dmc1 protein sequence data obtained to evaluate their utility as phylogenetic markers. We determined that evolutionary relationships of five of the six currently recognized eukaryotic supergroups are supported with Bayesian phylogenetic analyses. Using this dataset, we also identified ten amino acid residues that are highly conserved among Rad51 and Dmc1 protein sequences and, therefore, are likely to confer protein-specific functions. Due to the distributions of these residues, they are likely to have been present in the Rad51 and Dmc1 proteins of the LECA. To address an important issue with the gene inventory method of scientific inquiry, we developed a heuristic metric for determining whether apparent gene absences are due to limitations of the sequence search regimen or represent true losses of genes from genomes. We collected RNA polymerase I (Pol I), Replication Protein A (RPA), and DNA strand exchange (SE) sequence data from 47 diverse eukaryotes. We then compared the numbers of apparent absences to a single measure of protein sequence length and sequence conservation (Smith-Waterman pairwise alignment (S-W) scores) obtained by comparing yeast and human protein sequence data. Using Poisson correlation regression to analyze the Pol I and RPA subunit datasets, we confirmed that S-W scores and apparent gene absences are correlated. We also determined that genes encoding products that are critical for interhomolog SE in model organisms (Rad52, Rad51, Dmc1, Rad54, and Rdh54) have been lost frequently during eukaryotic evolution. Saccharomyces cerevisiae null rad52, dmc1, rad54, and rdh54 mutant phenotypes are suppressed by rad51 overexpression or mutation. If rad51 overexpression or mutation affects other eukaryotes in a similar fashion, this phenomenon may account for frequent losses of genes whose products are critical for the completion of meiosis in model organisms. Finally, we place this work into greater context with a review of hypotheses for the selective forces and mechanisms that resulted in the origin of meiosis. The review and the data presented in this thesis provide the basis for a model of the origin of meiotic genes in which meiosis arose from mitosis by large-scale gene duplication, following a preadaptation that served to reduce increased numbers of chromosomes (from diploid to haploid) caused by erroneous eukaryotic cell-cell fusions.