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p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

At the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA. Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage. / text

Meiotic recombination involves the exchange of DNA between two homologous chromosomes, forming cross-overs and gene conversion events. The cross-over process is important for the proper segregation of chromosomes during meiosis, and drives genetic diversity. Human hotspots are enriched for a 13-bp motif, CCNCCNTNNCCNC; a close match to this motif occurs in about 40% of our cross-over hotspots. A DNA binding protein called PRDM9, having histone trimethyltransferase (H3K4me3) activity, binds the motif and is becoming established as a major determinant of recombination hotspots (narrow regions with high cross-over activity). This research aimed to understand the mechanisms involved in promoting PRDM9 binding to its target sites, and subsequently, initiating cross-over hotspot activity. We first explored the relationship between PRDM9 binding and DNA sequence, to directly confirm whether PRDM9 binds to the 13-bp hotspot motif using in-vitro gel-shift assays, and found that it does bind sequence specifically to the canonical 13-mer motif. PRDM9 is able to bind the motif in a highly selective manner, with certain single base pair changes abolishing binding. However, we observe that it is also able to tolerate degeneracy in its binding sites, as demonstrated by strong in-vitro binding to degenerate versions of the 13-bp motif. Hence, these results confirmed that PRDM9 is able to directly bind to the 13-bp hotspot motifs, and given that it can also tolerate degeneracy, this raised the question of why PRDM9 is able to bind only a subset of all such potential binding sites in the genome. To address this, a ChIP-seq analysis was performed to identify genome wide binding sites for PRDM9. This information also helped us to characterise binding sites and investigate if factors such as the local chromatin environment play a role in specifying PRDM9 binding tar- gets and hotspot formation. We were able to identify over 170,000 PRDM9 binding sites in the genome. Surprisingly, these binding sites were also enriched in promoter regions, however, bound sites in these regulatory regions showed low recombination activity. We found that PRDM9 is able to confer the H3K4me3 mark on all bound sites, even those without a pre-existing H3K4me2 mark. We also investigated the role of other chromatin related marks on PRDM9 binding and found that binding occurs in chromatin accessible, but nucleosome rich regions, whereas heterochromatin regions tend to inhibit binding. Further, for hotspot formation, it was seen that less chromatin accessible, nucleosome dense regions away from transcribed sites, are preferred. Hotspots tend to avoid regions marked by transcription activating histone modifications, however, these regions do not appear to inhibit PRDM9 binding itself. These results show how PRDM9 binding in the genome is dependent on both primary DNA sequence and the surrounding epigenetic factors. Together these factors promote binding and, with additional downstream factors, positioning of hotspot locations in the human genome.

Meiotic recombination promotes genetic variation by reciprocal exchange of genetic material producing novel allelic combinations that influence important agronomic traits in crop plants. Therefore, harnessing meiotic recombination has the potential to accelerate crop improvement via classical breeding. Numerous genes involved in crossover formation have been identified in model systems. For example, SPO11 mediates generation of meiotic DNA double-strand breaks (DSBs) across all eukaryotes, which may be repaired as crossovers. However, downstream regulators of recombination remain to be identified, including those with species-specific roles. To isolate crossover frequency modifiers I performed a high-throughput forward genetic screen using EMS mutagenesis of Arabidopsis carrying a fluorescent crossover reporter line called 420. The primary screen isolated nine mutants from ~3,000 scored individuals that showed significantly higher (high crossover rate, hcr) or lower (low crossover rate, lcr) crossover frequency, including a new fancm allele. Four mutants (hcr1, hcr2, hcr3 and lcr1) were mapped by sequencing and candidate genes identified. The hcr1 mutation was confirmed as being located within the PROTEIN PHOSPHATASE X-1 (PPX-1) gene, using isolation of an independent allele and complementation studies. Similarly, the lcr1 mutation was confirmed to be within the gene TBP-ASSOCIATED FACTOR 4B (TAF4B). Using immunocytological staining I observed that hcr1 did not show changes in DSB-associated foci (RAD51), but it did show a significant increase in crossover-associated MLH1 foci. The hcr1 mutation increases crossovers mainly in the sub-telomeric chromosome regions, which remain sensitive to crossover interference. Also the genetic interaction between the hcr1 and fancm mutations is additive. These results support a model where PPX- 1 acts to limit recombination via the Class I interfering CO pathway, downstream of DSB formation. In summary, this genetic screen has led to discovery of novel genes that regulate meiotic recombination and their functional characterization may find utility in crop breeding programs.

La recombinaison homologue au cours de la méiose est un événement essentiel pour la ségrégation fidèle des chromosomes homologues, et contribue à la production de la diversité génétique. La recombinaison méiotique est initiée par l'induction de cassures double brin d'ADN (CDB), catalysée par SPO11, à des régions spécifiques du génome appelés points chauds. Récemment, il a été montré que PRDM9 est un déterminant majeur des points chauds de recombinaison chez la souris et l'homme. PRDM9 contient un domaine PR/SET avec une activité d'histone méthyltransférase, un domaine de liaison à l'ADN constitué d'une série de doigts de zinc en tandem, et des domaines prédit pour être impliqué dans des interactions protéine-protéine. Notre modèle de travail récent place PRDM9 comme un élément clé pour l'initiation de la recombinaison méiotique: PRDM9 se lie à l'ADN via le domaine à doigts de zinc, et modifie localement la structure de la chromatine. Grâce à un processus encore inconnu, SPO11 est recruté à proximité des sites de liaison de PRDM9, où il catalyse la formation de CDB. Le but de ma thèse était de répondre à la question : comment PRDM9 recrute-t-elle la machinerie CDB aux points chauds ? Pour mieux comprendre ce mécanisme, je me suis attaché à la caractérisation des protéines interagissant avec PRDM9. Les protéines interagissant potentiellement avec PRDM9 ont été identifiées, par criblage double hybrides dans la levure avec des banques d'ADNc issues de testicules, et par purification par affinité-spectrométrie de masse des complexes PRDM9. La cartographie par double hybride avec des formes tronquées de PRDM9 a révélé que le domaine KRAB atypique de PRDM9 joue un rôle clé dans les interactions protéine-protéine. Les protéines identifiées comprennent CXXC1, un composant évolutivement conservé du complexe SET1-COMPASS, et HELLS qui est indispensable à la progression de la méiose I chez la souris. J’ai montré que ces deux protéines sont exprimées au cours de la spermatogenèse chez la souris. Puisque Spp1, l'orthologue chez S. cerevisiae de CXXC1, est connu pour servir de médiateur de recrutement de la machinerie de formation des CDB aux sites de CBD, l'interaction entre PRDM9 et CXXC1 pourrait refléter la conservation de la fonction méiotique de Spp1 chez la souris. / Meiotic homologous recombination is an essential event for faithful segregation of homologous chromosomes, and contributes to production of genetic diversity. Meiotic recombination is initiated by the induction of programmed DNA double strand breaks (DSBs), which are catalyzed by SPO11, at specific regions of the genome called hotspots. Recently, PRDM9 was reported as a major determinant of recombination hotspots in mouse and human. PRDM9 contains a PR/SET domain with histone methyltransferase activity, a zinc-finger array, and putative domains for protein-protein interactions. Our recent working model involves PRDM9 as a key component for the initiation of meiotic recombination: PRDM9 binds DNA via the zinc-finger array, and modifies chromatin structure locally. Through an unknown process, SPO11 is recruited and catalyzes DSB formation near PRDM9-bound sites. The aim of my thesis was to address the question: how does PRDM9 recruit DSB machinery to hotspots. To gain insight into this mechanism, I focused on characterization of PRDM9-interacting proteins. Potential interactors of PRDM9 were identified by yeast two hybrid (Y2H) screens with testis cDNA libraries and by affinity purification-mass spectrometry of PRDM9 complexes. Further Y2H assays with truncated derivatives of PRDM9 revealed that the atypical KRAB domain of PRDM9 plays a key role in protein-protein interactions. The identified proteins include CXXC1, a component of the evolutionarily conserved SET1-COMPASS complex, and HELLS, which is indispensable for progression of meiotic prophase I in mouse. Both proteins were found to be expressed during mouse spermatogenesis. Since Spp1, the S.cerevisiae orthologue of CXXC1, is known to mediate tethering of DSB sites to DSB machinery, the interaction between PRDM9 and CXXC1 might imply potential conservation of the Spp1 function in mouse meiosis.

La recombinaison homologue est un processus conservé chez les eucaryotes. Au cours de la méiose,ce mécanisme est essentiel à la formation des crossing-overs, eux-mêmes essentiels à la bonne ségrégation des chromosomes homologues. La recombinaison méiotique est assurée par l’action combinée de facteurs mitotiques et méiotiques. La protéine MEIOB a récemment été identifiée et caractérisée comme étant essentielle à la réparation des cassures double brin de l’ADN au cours de la méiose. MEIOB est un paralogue de RPA1, la grande sous-unité du complexe RPA qui est un complexe de liaison à l’ADN simple brin ubiquitaire et composé de RPA1, RPA2 et RPA3. MEIOB peut interagir avec SPATA22 et RPA2. Cette observation suggère que MEIOB, SPATA22 et RPA pourraient agir ensemble au cours de la recombinaison méiotique. En se basant sur l’homologie de structure entre MEIOB, SPATA22 et les sous-unités de RPA, nous avons caractérisé les modalités et le rôle de leur interaction. Nous avons montré que MEIOB et SPATA22 interagissent grâce à leur domaines OB-folds C-terminaux à l’image de RPA1 et RPA2 et que MEIOB et SPATA22 coopèrent pour interagir avec le complexe RPA. Par microscopie électronique, nous avons mis en évidence que la présence de MEIOB-SPATA22 induit une forte condensation du filament RPA ADN simple brin. Nous avons également montré par immunofluorescence sur chromosomes méiotiques murins que l’hélicase BLM accumule sur les axes chromosomiques et que cette accumulation est corrélée avec l’élimination de la recombinase DMC1 des cassures méiotiques non-réparées, en absence de MEIOB. Enin, nous avons mis en évidence par microscopie à haute résolution que l’absence de MEIOB favorise une distribution anormale des protéines recombinases. Nos résultats suggèrent que MEIOB, SPATA22 et RPA collaborent pour assurer l’intégrité des intermédiaires de recombinaison méiotiques au cours de l’invasion d’un brin homologue. / Homologous recombination is a conserved process among eukaryotes. During meiosis, thismechanism is essential to the formation of crossovers and thus for the proper segregation of chromosomes. Meiotic recombination is ensured by the combined action of mitotic and meiotic factors. MEIOB has been recently identiied and shown to be essential to the repair of meiotic DNA double-strand breaks. MEIOB is aparalog of RPA1, the large subunit of RPA, which is a ubiquitous ssDNA-binding trimeric composed ofRPA1, RPA2 and RPA3. MEIOB has been shown to interact with SPATA22 and RPA2. This observation suggested that MEIOB, SPATA22 and RPA may work together. Based on the homology existing betweenstructural domains of MEIOB, SPATA22 and the RPA subunits, we deciphered the modality and the role oftheir interactions. We show that MEIOB and SPATA22 interact through their C-terminal OB domains like RPA1 and RPA2 and cooperate to interact with the RPA complex. Using Transmission Electron Microscopy,we evidenced that the presence of MEIOB/SPATA22 induces a strong compaction of the RPA/ssDNAilament. Immunofluorescent microscopy performed on murin meiotic chromosomes revealed that in theabsence of MEIOB, the BLM helicase accumulates on chromosomes axis and correlates with the eviction ofthe DMC1 recombinase from unrepaired meiotic breaks. Finally, we show that the absence of MEIOB favorsabnormal recombinase distribution observed by SIM microscopy. Together, our results evidence thatMEIOB, SPATA22 and RPA act together to insure the integrity of recombination intermediates during strandinvasion.

Previous evidence has shown that the simple sequences microsatellites and poly-purine/poly-pyrimidine tracts (PPTs) could be both a cause, and an effect, of meiotic recombination. The causal link between simple sequences and recombination has not been much explored, however, probably because other evidence has cast doubt on its generality, though this evidence has never been conclusive. Several questions have remained unanswered in the literature, and I have addressed aspects of three of them in my thesis. First, what is the scale and magnitude of the association between simple sequences and recombination? I found that microsatellites and PPTs are strongly associated with meiotic double-strand break (DSB) hotspots in yeast, and that PPTs are generally more common in human recombination hotspots, particularly in close proximity to hotspot central regions, in which recombination events are markedly more frequent. I also showed that these associations can't be explained by coincidental mutual associations between simple sequences, recombination and other factors previously shown to correlate with both. A second question not conclusively answered in the literature is whether simple sequences, or their high levels of polymorphism, are an effect of recombination. I used three methods to address this question. Firstly, I investigated the distributions of two-copy tandem repeats and short PPTs in relation to yeast DSB hotspots in order to look for evidence of an involvement of recombination in simple sequence formation. I found no significant associations. Secondly, I compared the fraction of simple sequences containing polymorphic sites between human recombination hotspots and coldspots. The third method I used was generalized linear model analysis, with which I investigated the correlation between simple sequence variation and recombination rate, and the influence on the correlation of additional factors with potential relevance including GC-content and gene density. Both the direct comparison and correlation methods showed a very weak and inconsistent effect of recombination on simple sequence polymorphism in the human genome.Whether simple sequences are an important cause of recombination events is a third question that has received relatively little previous attention, and I have explored one aspect of it. Simple sequences of the types I studied have previously been shown to form non-B-DNA structures, which can be recombinagenic in model systems. Using a previously described sodium bisulphite modification assay, I tested for the presence of these structures in sequences amplified from the central regions of hotspots and cloned into supercoiled plasmids. I found significantly higher sensitivity to sodium bisulphite in humans in than in chimpanzees in three out of six genomic regions in which there is a hotspot in humans but none in chimpanzees. In the DNA2 hotspot, this correlated with a clear difference in numbers of molecules showing long contiguous strings of converted cytosines, which are present in previously described intramolecular quadruplex and triplex structures. Two out of the five other hotspots tested show evidence for secondary structure comparable to a known intramolecular triplex, though with similar patterns in humans and chimpanzees. In conclusion, my results clearly motivate further investigation of a functional link between simple sequences and meiotic recombination, including the putative role of non-B-DNA structures.

La protéine Mlh1 eucaryote est un acteur central de la voie de réparation des mésappariements (MMR). Chez la levure, Mlh1 forme un hétérodimère via sa région C-terminale avec les endonucléases Pms1 et Mlh3. La région C-terminale de Mlh1 est également en interactions avec l’exonucléase Exo1 du MMR et deux protéines Ntg2 et Sgs1 qui sont impliquées dans d’autres voies de réparation. Dans un premier temps, nous avons identifié et caractérisé le site d’interaction de Mlh1 avec les protéines Exo1, Ntg2 et Sgs1, qui utilisent un même motif de 5 acides aminés, (R/K)SK(Y/F)F appelé motif MIP pour Mlh1 Interacting Protein. Nous avons montré que ces 3 protéines interagissent en un même site, appelé site S2. Nous avons identifié 10 positions de Mlh1 impliquées dans le site S2 et caractérisé par microcalorimétrie, une affinité micromolaire entre des peptides contenant le motif MIP et la région C-terminale de Mlh1. Nous avons montré que les protéines EXO1 et BLM humaines qui possèdent également un motif MIP, interagissent spécifiquement avec MLH1 humain par ce motif. Dans un second temps, nous avons résolu la structure cristallographique à 2.6Å de la région C-terminale de l’hétérodimère Mlh1*Pms1. Le site d’hétérodimèrisation présente une surface d’interaction supérieure à celle observée dans les homodimères de MutL bactériens. La structure résolue confirme le rôle des 10 acides aminés de Mlh1 identifiés lors de la caractérisation du site S2. La structure du site endonucléase de Pms1 révèle la présence de deux atomes de zinc chelatés par 5 acides aminés de Pms1 et le dernier acide aminé de la protéine Mlh1, la cystéine C769. Cette première structure d’une région C-terminale d’un complexe Mlh1*Pms1 eucaryote permet d’analyser la position des nombreux mutants ponctuels de MLH1 humain associés à des cancers du côlon HNPCC. / Eucaryotic Mlh1 is a core component of mismatch repair pathway (MMR). In yeast organisms, Mlh1 forms heterodimer with its C-terminal region with endonucleases Pms1 and Mlh3. The C-terminal region of Mlh1 is also involved in interactions with MMR exonuclease Exo1 and two proteins, Ntg2 and Sgs1, which are involved in other DNA repair pathways. First, we identified and charaterised the interaction site between Mlh1 and proteins Exo1, Ntg2, and Sgs1, that share the same motif of 5 amino acids, (R/K)SK(Y/F)F, named MIP box for Mlh1 Interacting Protein. We showed that these 3 proteins bind to the same site, named site S2. 10 positions of Mlh1 important for interactions on site S2 were identified and a micromolar affinity was measured by calorimetry between the C-terminal region of Mlh1 and peptides containing a MIP box. We showed that human EXO1 and BLM specifically with human MLH1 through their MIP box. Secondly, we solved the X-ray structure of the C-terminal region of Mlh1*Pms1 heterodimer at 2.6Å. The structure shows that the surface buried upon heterodimerisation is higher in eucaryotes than in MutL homodimers. The structure confirms the overall structure of the site S2 predicted in the first part of this study. The endonuclease site of Pms1 presents in the crystal two zinc atoms that are bound by five Pms1 residues and the last residue of Mlh1 chain, cystein C769. This structure represents the first image of the C-terminal region of an eucaryote Mlh1*Pms1 heterodimer. It allows localizing the positions of human MLH1 mutants associated with colon cancers named HNPCC.