<p>Cancer cells often have elevated frequencies of chromosomal aberrations, and it is likely that loss of genome stability is one driving force behind tumorigenesis. Deficiencies in DNA replication, DNA repair, or cell cycle checkpoints can all contribute to increased rates of chromosomal duplications, deletions and translocations. The human ATM and ATR proteins are known to participate in the DNA damage response and DNA replication checkpoint pathways and are critical to maintaining genome stability. The <italic>Saccharomyces cerevisiae</italic> homologues of ATM and ATR are Tel1p and Mec1p, respectively. Because Tel1p and Mec1p are partially functionally redundant, loss of both Tel1p and Mec1p in haploid yeast cells (<italic>tel1 mec1</italic> strains) results in synergistically elevated rates of chromosomal aberrations, including terminal duplications, chromosomal duplications, and telomere-telomere fusions. To determine the effect of Tel1p and Mec1p on chromosome aberrations that cannot be recovered in haploid strains, such as chromosome loss, I investigated the phenotypes associated with the <italic>tel1 mec1</italic> mutations in diploid cells. In the absence of induced DNA damage, <italic>tel1 mec1</italic> diploid yeast strains exhibit extremely high rates of aneuploidy and chromosome rearrangements. There is a significant bias towards trisomy of chromosomes II, VIII, X, and XII, whereas the smallest chromosomes I and VI are commonly monosomic. </p> <p> The telomere defects associated with <italic>tel1 mec1</italic> strains do not cause the high rates of aneuploidy, as restoring wild-type telomere length in these strains by expression of the Cdc13p-Est2p fusion protein does not prevent cells from becoming aneuploid. The <italic>tel1 mec1</italic> diploids are not sensitive to the microtubule-destabilizing drug benomyl, nor do they arrest the cell cycle in response to the drug, indicating that the spindle assembly checkpoint is functional. The chromosome missegregation phenotypes of <italic>tel1 mec1</italic> diploids mimic those observed in mutant strains that do not achieve biorientation of sister chromatids during mitosis. </p> <p> The chromosome rearrangements in <italic>tel1 mec1</italic> cells reflect both homologous recombination between non-allelic Ty elements, as well as non-homologous end joining (NHEJ) events. Restoring wild-type telomere length with the Cdc13p-Est2p fusion protein substantially reduces the levels of chromosome rearrangements (terminal additions and deletions of chromosome arms, interstitial duplications, and translocations). This result suggests that most of the rearrangements in <italic>tel1 mec1</italic> diploids are initiated by telomere-telomere fusions. One common chromosome rearrangement in <italic>tel1 mec1</italic> strains is an amplification of sequences on chromosome XII between the left telomere and rDNA sequences on the right arm. I have termed this aberration a "schromosome." Preliminary evidence indicates that the schromosome exists in the <italic>tel1 mec1</italic> cells as an uncapped chromosome fragment that gets resected over time.</p> / Dissertation

L'ADN de chaque cellule est constamment soumis à des stress pouvant compromettre son intégrité. Les bris double-brins sont probablement les dommages les plus nocifs pour la cellule et peuvent être des sources de réarrangements chromosomiques majeurs et mener au cancer s’ils sont mal réparés. La recombinaison homologue et la jonction d’extrémités non-homologues (JENH) sont deux voies fondamentalement différentes utilisées pour réparer ce type de dommage. Or, les mécanismes régulant le choix entre ces deux voies pour la réparation des bris double-brins demeurent nébuleux. Le complexe Mre11-Rad50-Xrs2 (MRX) est le premier acteur à être recruté à ce type de bris où il contribue à la réparation par recombinaison homologue ou JENH. À l’intersection de ces deux voies, il est donc idéalement placé pour orienter le choix de réparation. Ce mémoire met en lumière deux systèmes distincts de phosphorylation du complexe MRX régulant spécifiquement le JENH. L’un dépend de la progression du cycle cellulaire et inhibe le JENH, tandis que l’autre requiert la présence de dommages à l’ADN et est nécessaire au JENH. Ensembles, nos résultats suggèrent que le complexe MRX intègre différents phospho-stimuli pour réguler le choix de la voie de réparation. / The genome of every cell is constantly subjected to stresses that could compromise its integrity. DNA double-strand breaks (DSB) are amongst the most damaging events for a cell and can lead to gross chromosomal rearrangements, cell death and cancer if improperly repaired. Homologous recombination and non-homologous end joining (NHEJ) are the main repair pathways responsible for the repair of DSBs. However, the mechanistic basis of both pathways is fundamentally different and the regulation of the choice between both for the repair of DSBs remains largely misunderstood. The Mre11-Rad50-Xrs2 (MRX) complex acts as a DSB first responder and contributes to repair by both homologous recombination and NHEJ. Being at the crossroads of both DSB repair pathways, the MRX complex is therefore in a convenient position to influence the repair choice. This thesis unravels two distinct phosphorylation systems modifying the MRX complex and specifically regulating repair by NHEJ. The first relies on cell cycle progression and inhibits NHEJ, while the second requires the presence of DNA damage and is necessary for efficient NHEJ. Together, our results suggest a model in which the MRX complex would act as an integrator of phospho-stimuli in order to regulate the DSB repair pathway choice.

L'ADN de chaque cellule est constamment soumis à des stress pouvant compromettre son intégrité. Les bris double-brins sont probablement les dommages les plus nocifs pour la cellule et peuvent être des sources de réarrangements chromosomiques majeurs et mener au cancer s’ils sont mal réparés. La recombinaison homologue et la jonction d’extrémités non-homologues (JENH) sont deux voies fondamentalement différentes utilisées pour réparer ce type de dommage. Or, les mécanismes régulant le choix entre ces deux voies pour la réparation des bris double-brins demeurent nébuleux. Le complexe Mre11-Rad50-Xrs2 (MRX) est le premier acteur à être recruté à ce type de bris où il contribue à la réparation par recombinaison homologue ou JENH. À l’intersection de ces deux voies, il est donc idéalement placé pour orienter le choix de réparation. Ce mémoire met en lumière deux systèmes distincts de phosphorylation du complexe MRX régulant spécifiquement le JENH. L’un dépend de la progression du cycle cellulaire et inhibe le JENH, tandis que l’autre requiert la présence de dommages à l’ADN et est nécessaire au JENH. Ensembles, nos résultats suggèrent que le complexe MRX intègre différents phospho-stimuli pour réguler le choix de la voie de réparation. / The genome of every cell is constantly subjected to stresses that could compromise its integrity. DNA double-strand breaks (DSB) are amongst the most damaging events for a cell and can lead to gross chromosomal rearrangements, cell death and cancer if improperly repaired. Homologous recombination and non-homologous end joining (NHEJ) are the main repair pathways responsible for the repair of DSBs. However, the mechanistic basis of both pathways is fundamentally different and the regulation of the choice between both for the repair of DSBs remains largely misunderstood. The Mre11-Rad50-Xrs2 (MRX) complex acts as a DSB first responder and contributes to repair by both homologous recombination and NHEJ. Being at the crossroads of both DSB repair pathways, the MRX complex is therefore in a convenient position to influence the repair choice. This thesis unravels two distinct phosphorylation systems modifying the MRX complex and specifically regulating repair by NHEJ. The first relies on cell cycle progression and inhibits NHEJ, while the second requires the presence of DNA damage and is necessary for efficient NHEJ. Together, our results suggest a model in which the MRX complex would act as an integrator of phospho-stimuli in order to regulate the DSB repair pathway choice.