Dans la plupart des eucaryotes, il n’existe qu’une seule région centromérique par chromosome et celle-ci est capable d’être liée au fuseau mitotique via le complexe du kinétochore. Dans ce contexte, la présence de deux centromères est un défi pour une séparation normale. Au cours de la mitose, la capture des deux centromères de la même chromatides vers les pôles opposés génère un pont d’anaphase résultant en une rupture entre les centromères. Les extrémités libérées peuvent être fusionnées bout à bout recréant ainsi un dicentrique. Le chromosome entre alors dans un cycle de Rupture Cassure Pont, capable quelques cycles d’entrainer des modifications profondes du nombre de copies de gène qui peuvent contribuer à l'oncogenèse et résistance à la chimiothérapie. Malgré son importance, le mécanisme de rupture reste pour une grande partie inexploré. Ce projet permet l’analyse de la rupture des chromosomes dicentriques en utilisant le modèle de la levure bourgeonnante, Saccharomyces cerevisiae. Nous utilisons des souches dicentriques conditionnelles dans lequelles un chromosome, portant un centromère conditionnel sous le contrôle de deux promoteurs inductibles au galactose, est fusionné à un autre chromosome natif par recombinaison homologue. Nous avons observé que les chromosomes dicentriques ont tendance à casser dans le voisinage des deux centromères. La région de la rupture se répand sur ~ 30 kb vers l'autre centromère. Une insertion d’un fragment d’ADN 1-kb possédant un centromère ectopique dans un chromosome avec un centromère conditionnelle établit un point chaud d’environs 30 kb indiscernables des points chauds à centromères natifs. En outre, la taille de zone de rupture n’est pas corrélée à la distance intercentromerique (des intervalles de 30-600 kb ont été testés). Cela indique que la plus forte propension à rompre est une conséquence de la structure ou de la fonction des centromères et est sans rapport avec les séquences environnantes des chromosomes. Il est encore difficile de savoir si la rupture aux centromères a une fonction physiologique, mais nous pouvons supposer que ce point chaud peut favoriser les réarrangements d'ADN dans ces régions permettant ainsi l’inactivation du centromère et donc le retour à un caryotype stable. Globalement dans la S.cerevisiae, les dicentriques cassent dans les régions péricentromériques ou dans les fusions de télomères quand ils sont présents. Fait intéressant, les séquences télomériques internes, à savoir les répétitions TG₁₋₃, établissent plusieurs points chauds de rupture à une fréquence similaire. En perspective, il serait intéressant d'aborder les questions suivantes : 1) Quelles sont les caractéristiques qui rendent une région plus sujette à la casse ? 2) Quelles sont les positions de rupture au niveau des nucléotides ? 3) Existe-t-il un contrôle de la cassure des chromatides exercé dans la cellule ? 4) Quelle peut être la fonction biologique des points chauds de cassures ? / In most eukaryotes, there is one defined centromeric region per chromosome that links it to the spindle apparatus via the kinetochore complex. In this context, the presence of two centromeres is a challenge for an accurate segregation. During mitosis, the capture of the two centromeres of the same chromatid to opposite poles generates anaphase bridges that results in breakage between the centromeres. The released ends can be fused end-to-end thus recreating dicentric. It enters breakage-fusion-bridge cycles that, in multiple rounds, can result in large gene copy number alterations that can contribute to oncogenesis and chemotherapy resistance. Despite of its significance, the mechanism of breakage remains for a large part unexplored. This project adresses the dicentric breakage using a budding yeast, Saccharomyces cerevisiae. We use conditional dicentric strains, where a chromosome, bearing a conditional centromere under the control of two galactose-inducible promoters, is fused to another native chromosome by homologous recombination. We observed that dicentric chromosomes tend to break in the vicinity of the two centromeres. The breakage region spreads over ~30 kb towards the other centromere. An insertion of a 1-kb ectopic centromere in a chromosome with a conditional centromere establishes a ~30 kb hot spot indistinguishable from the hot spots at native centromeres. Furthermore, the size of breakage region is unrelated to an intercentromeric distance (30-600 kb intervals were tested). This indicates that the higher propensity to break is a consequence of centromere structure or function and is unrelated to the native surrounding sequences. It is yet unclear whether breakage at centromeres has a physiological function but we can speculate that this hot spot may favour local DNA rearrangements that result in centromere inactivation and thus the return to a stable karyotype. Overall in budding yeast, dicentrics break at pericentromeric regions or at the telomere fusions when they are present. Interestingly, internal telomeric sequences, i.e. TG₁₋₃ repeats, establish several breakage hot spots with a similar frequency. In perspective, it would be interesting to address the following questions: 1) What are features that make a region more prone to breakage? 2) What are the positions of breakage at nucleotide level? 3) Is there a coordination of dicentric chromatid breakage? 4) What can be the biological function of dicentric breakage hot spots?

Chromosomal fragile sites are specific loci associated with a high frequency of breakage and recombination. A cell's ability to repair and/or replicate through a lesion is prerequisite to the maintenance of genomic stability. An improved understanding of fragile site biology and its contribution to replication defects and genomic instability is critical for prevention, intervention, and diagnosis of genetic diseases such as cancer. This work seeks to identify and characterize both trans and cis fragile sites associated elements involved in instability onset and progression. An array of Saccharomyces cerevisiae isogenic DNA repair deficient mutants were utilized to identify genes contributing to the stability or instability of a natural fragile site ~ 403 kb from the left telomere on chromosome VII. Findings suggest that the RAD52 epistasis group, the MRX complex, non-homologous end-joining (NHEJ) pathways, MUS81 and SGS1 helicases, translesion polymerases, and a majority of the post replication repair (PRR) proteins are all required for faithful replication of the 403 fragile site and likely other fragile sites as well. In contrast I found that MMS2, previously thought to be specific to the PRR pathway, is required to prevent the fusion of repetitive elements within the 403 site. mgs1 (homolog of the human Werner helicase interacting protein, WHIP) and pol3-13 (a subunit of the DNA polymerase delta) mutants also exhibited reduced instability in checkpoint deficient cells. These findings suggest previously uncharacterized function of Mgs1, Pol3 and Mms2 in regulation of genome regions at risk of replication damage. We further find the presence of inverted repeats (IR) are sufficient to induce instability. Two IR's proximal to the 403 site consistently fuse to generate acentric and dicentric chromosomes involving the 403 fragile site and a newly identified site on chromosome VII as well. The frequency of fusion events is aggravated by chromatin traffic stressors such as tRNA transcription induced fork stalling and replisome termination regions.

Large-scale changes are common in genomes, and are often associated with pathological disorders. In the work presented in this dissertation, I provide insights into how inverted repeat sequences in budding yeast fuse during replication. Fusion leads to the formation of dicentric chromosomes, a translocation, and other chromosomal rearrangements.Using extensive genetics and some molecular analyses, I demonstrate that dicentric chromosomes are key intermediates in genome instability of a specific chromosome in budding yeast. I provide three pieces of evidence that is consistent with this conclusion. First, I detect a recombination fusion junction that is diagnostic of a dicentric chromosome (using a PCR technique). Second, I show a strong correlation between the amount of the dicentric fragment and the frequency of instability of the entire chromosome. Third, I demonstrate that a mutant known to stabilize dicentric chromosomes suppress instability. Based on these observations, I conclude that dicentric chromosomes are intermediates in causing genome instability in this system.Next, we demonstrate that fusion of inverted repeats is general. Both endogenous and synthetic nearby inverted repeats can fuse. Using genetics, I also show that many DNA repair and checkpoint pathways suppress fusion of nearby inverted repeats and genome instability. Based on our analysis, we propose a novel mechanism for fusion of inverted repeats that we term `faulty template switching.'Lastly, I discuss two genes that are necessary for fusion of nearby inverted repeats. I identified a mutant of the Exonuclease 1 (Exo1) and a mutant of anaphase inhibitor securin (Pds1) that suppress nearby inverted repeat fusion and genome instability. Studies of Exo1 and Pds1 provide us with insights into the molecular mechanisms of fusion.Our finding that nearby inverted repeats can fuse to form dicentric chromosomes that lead to genome instability may have great implications. The generality of this fusion reaction raises the possibility that dicentric chromosomes formed by inverted repeats can lead to genome instability in mammalian cells, and thereby contribute to a cancer phenotype.

Chromosomal rearrangements occur in all organisms and are important both in the evolution of species and in pathology. In this dissertation I show that in Saccharomyces cerevisiae, or budding yeast, one type of chromosomal rearrangement occurs when inverted repeats fuse, likely during DNA replication by a novel mechanism termed "faulty template switching". This fusion can lead to the formation of either a dicentric or acentric chromosome, depending on the direction of the replication fork. Dicentric chromosomes are inherently unstable due to their abnormal number of centromeres, and thus undergo additional chromosomal rearrangements and chromosome loss.

The integrity of the genome relies on the maintenance of chromosomes, the structural embodiment of the genetic material. Disruption of chromosome replication can lead to extensive genomic rearrangements, spanning kilobase (Kb) to megabase (Mb) regions. Some chromosome rearrangements are inherently dynamic, beginning as a single unstable rearrangement from which multiple rearrangements emerge. The rare formation and transient behavior of unstable chromosomes renders their study challenging. Here I characterize the genetic ontogeny of unstable chromosomes in a budding yeast model, from initial replication error to unstable chromosome formation to their resolution. I find that the initial error often arises in or near the telomere and frequently forms unstable chromosomes that later resolve to an internal "collection site" in the middle of the chromosome. The initial telomere-proximal unstable chromosome is increased in cells mutant for telomerase, the Tel1 checkpoint kinase and even the Rad9 checkpoint protein, with no known telomere-specific function. Defects in Tel1 and the Rrm3 DNA helicase, or the Tel1-MRX complex and 9-1-1 checkpoint clamp, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. I performed a candidate genetic screen of instability in telomere maintenance and DNA damage response (DDR) proteins to characterize the interplay of pathways regulating senescence and genomic instability. Collectively, my results suggest that unstable chromosomes form in or near damaged telomeres, independently of end degradation (Exo1-independent), by either nonhomologous end joining (partially Lig4-dependent) or by faulty template switch during replication (Lig4- and Rad52-independent). The telomere-proximal unstable chromosomes then rearrange further to the middle of the chromosome. These results implicate telomere replication errors as a common source of widespread genomic changes and make substantial progress to our understanding of the initiation and fate of unstable chromosomes in the eukaryotic genome.