Thesis advisor: Clare O'Connor / Telomeres are repeated sequences at the ends of chromosomes, which promote chromosome stability by preventing the loss of necessary nucleotides from the DNA with successive rounds of replication. Telomeres are elongated by the enzyme telomerase, which has both a protein component and an RNA component. In the yeast Saccharomyces cerevisiae, the TLC1 gene encodes the RNA component of the enzyme. Telomerase RNA interacts with several proteins to perform its function, including the Ku protein, which binds to the end of the DNA and helps to recruit telomerase to the chromosome thereby facilitating the lengthening of chromosome ends. Ku interacts with telomerase RNA at the site of a 48-nucleotide stem-loop on the RNA's structure. Previous experiments have shown that yeast strains engineered to carry two copies of the TLCI gene exhibit higher levels of telomerase RNA than those that have only one copy of the gene. Also, a yeast strain carrying a copy of the mutant tlc1Δ48 gene, which contains a deletion of the 48-nucleotide stem-loop, contains lower levels of telomerase RNA than a strain with the wild type TLC1 gene. This series of experiments is investigating whether the copy number of the telomerase RNA gene affects the elongation of telomeres in S. cerevisiae. In order to determine this effect, the de novo telomere addition of four strains was examined, as were the native telomere lengths of these strains. The assay indicated that the efficiency of telomere elongation was unchanged by increasing the copy number of the wild type gene but was increased upon increasing the copy number of the mutant gene. Analysis of the native telomere lengths showed that increasing the copy number of either the wild type or the mutant gene allowed the cells to maintain their telomeres at a longer length. / Thesis (BS) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.

Les cassures double-brins d’ADN (CDBs) constituent une menace pour la viabilité cellulaire et l’intégrité du génome puisque l’absence de la réparation d’une CDB pourrait conduire à la mort cellulaire. En plus de la réparation par jonction d’extrémités nonhomologues (NHEJ) en phase G1 et de la recombinaison homologue (RH) en phase S et G2, les CDBs peuvent être réparées par l’ajout de télomères par l’action de la télomérase; un phénomène qui s’appelle l’ajout de télomères de novo. Ce phénomène pourrait mettre en danger la stabilité génomique parce qu’il engendre, dans la plupart des cas, une perte du bras chromosomique du fragment non-centromérique. En conséquence, ceci engendre soit une perte de l’hétérozygotie (LOH) dans les cellules diploïdes ou la mort cellulaire dans les cellules haploïdes. Dans le but d’empêcher la formation de télomères de novo, la cellule possède des mécanismes et des voies qui préviennent l’action inappropriée de la télomérase à des CDBs. Une des principales questions dans le domaine est de comprendre comment la cellule inhibe l’ajout de télomères de novo par la télomérase en favorisant la réparation des CDBs par les autres voies (NHEJ et la RH).Dans ce projet, nous utilisons la technique d’hybridation in situ en fluorescence (FISH) sur le facteur limitant de la télomérase, l’ARN TLC1 de la levure S. cerevisiae. Nous avons pu montrer que l’ARN TLC1 fait un trafic intranucléaire durant le cycle cellulaire des cellules sauvages. En phase G1/S, l’ARN TLC1 adopte une localisation nucléoplasmique avec les télomères, alors qu’il s’accumule au nucléole en phase G2/M. Nous avons fait l’hypothèse que l’accumulation de l’ARN TLC1 au nucléole en G2/M pourrait réduire la compétition entre la RH, qui est exclusivement nucléoplasmique, et la télomérase pour la réparation des CDBs. Pour tester cette hypothèse, nous avons employé la bléomycine (blm), un composé chimique générant des CDBs, pour traiter des cellules sauvages ou déficientes de la RH par la délétion du gène RAD52. Nous avons observé que l’ARN TLC1 conserve une localisation nucléolaire dans les cellules sauvages traitées par la blm en phase G2/M, alors que dans lescellules délétées de RAD52 exposées à la blm, l’ARN TLC1 se localise maintenant au nucléoplasme et s’associe partiellement aux sites de cassures. De plus, nous avons trouvé que l’accumulation nucléoplasmique de l’ARN TLC1 dans les cellules délétéées de RAD52 traitées à la blm, dépend de la voie de dommage à l’ADN (MRX, ATM/Tel1 et ATR/Mec1) et de la sumoylation par la SUMO E3ligase, Siz1. Plus particulièrement, l’association de la télomérase à des CDBs dépend de son interaction avec Cdc13, une protéine qui recrute la télomérase aux télomères. D’une manière surprenante, nous avons observé une accumulation rapide de Cdc13 à des CDBs en absence de Rad52, bien que nos résultats suggèrent que Rad52 empêche l’accumulation de l’ARN TLC1 au nucléoplasme par l’inhibition de l’accumulation de Cdc13 aux sites de cassures. L’ensemble de nos résultats ont mis en évidence que la télomérase est normalement exclue des sites de la réparation d’ADN. Cependant, en absence d’une voie fonctionnelle de la RH, la télomérase se localise du nucléole au nucléoplasme et s’accumule partiellement à des CDBs d’une manière dépendante de Cdc13 et Siz1. / DNA double-strand breaks (DSB) constitute a threat to genome integrity and cell survival if they are not repaired. In addition to canonical DNA repair systems such as nonhomologous end joining (NHEJ) in G1 and homologous recombination (HR) in S and G2 phases, DSBs can also be repaired by addition of new telomeres by telomerase. This phenomenon is referred to as telomere healing or de novo telomere addition. This process threatens genome stability since it results in chromosome arm loss, which could be lethal in haploid cells and lead to loss of heterozygosity (LOH) in diploid cells. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. One of the questions driving this field is to understand how telomere addition by telomerase is inhibited and DSBs repair can be efficiently performed by canonical DSB repair (NHEJ and HR). In this project, we used fluorescent in situ hybridization (FISH) to detect the endogenous TLC1 RNA, which is the limiting component of telomerase of the budding yeast. Using this technique, we found that TLC1 RNA traffics inside the nucleus during the cell cycle of wild-type cells. In G1 and S phases, TLC1 RNA adopts a nucleoplasmic localization, which is related to its function in telomere elongation, while it accumulates in the nucleolus in G2/M. We hypothesize that the nucleolar accumulation of TLC1 RNA in G2/M may reduce the possibility that telomerase interferes with HR to repair DNA DSB, since HR is excluded from the nucleolus and occurs only in the nucleoplasm. To test this hypothesis, we treated wild-type and rad52 (HR deficient cells) with bleomycin, a radiomimetic agent that generates preferentially DSBs. Our results show that after induction of DSB with bleomycin, TLC1 RNA remains nucleolar in wild-type cells in G2/M, but accumulates in the nucleoplasm and colocalizes partially with DSBs sites in rad52 cells, suggesting that RAD52 inhibits the nucleoplasmic accumulation of TLC1 RNA in the presence of DSBs. Nucleoplasmic accumulation of TLC1 RNA after DSB induction requires the DNA damage pathway (MRX, ATM/Tel1 and ATR/Mec1), and the SUMO ligase E3 Siz1. Interestingly, association of TLC1 RNA with DSBs depends on the single-strand telomeric binding protein Cdc13, which rapidly accumulates at sites of DNA damage, while Rad52 suppresses this process by inhibiting Cdc13 accumulation at DSBs. These results suggest that telomerase is normally excluded from sites of DNA repair. In the absence of functional homologous recombination, telomerase leaves the nucleolus and accumulates partially at DSB in the nucleoplasm in a Cdc13- and Siz1-dependent manner.