When damaged DNA is detected during replication, a checkpoint delays the cell cycle to allow time for repair. Here I show that continually delaying the cell cycle in the G2/M phase of the cell cycle stabilizes the genome of Saccharomyces cerevisiae in both checkpoint proficient and deficient cells; a phenomenon I call slow cycle stabilization (SCS). SCS stabilizes the genome in cells defective for DNA damage response (DDR), spindle checkpoint, and telomere biology, as well as wild type (WT) cells. I verify SCS using genetic and chemical means and further substantiate SCS using three different Saccharomyces cerevisiae chromosome systems.

Mms1 and Mms22 are important mediators of genome stability in Saccharomyces cerevisiae. Deletion mutants of both genes display decreased viability in the presence of a number of known DNA damaging agents as well as agents that perturb DNA replication. Here I present a detailed analysis of Mms1 and Mms22 function in stabilizing DNA replication forks. I find that mms1∆ and mms22∆ strains accumulate spontaneous DNA damage and have an increased rate of mutation during a normal cell cycle. Additionally, treatment with genotoxic agents causes defects in induced recombination as well as inhibiting the ability of cells to efficiently recover from DNA damage. Genetic interaction data support a model where Mms1 and Mms22 function with components of the replication fork. Accordingly I observed that the enrichment of key replication fork proteins at regions proximal to replication origins is decreased in mms1∆ and mms22∆ cells during replication stress. Furthermore, I monitored DNA replication in wild-type and mms1∆ strains and found that mms1∆ strains exhibit irregular fork progression under conditions of replication stress. I therefore concluded that Mms1 and Mms22 function at replication forks. Lastly, I uncovered that Mms1 is regulated by Rtt101-dependent ubiquitin-mediated proteolysis. Mms1 degradation is likely necessary for its function as overexpression of Mms1 results in decreased cell viability both during a normal cell cycle, and during treatment with DNA damaging agents. I found that Mms1 and Mms22 operate at sites of DNA replication to promote the stability of the genome, especially under conditions of DNA damage and replication fork stress.

In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

<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

Telomeres are the physical ends of linear chromosomes in eukaryotes. Telomeres not only protect chromosome ends from being recognized as double-strand breaks but also maintain the chromosome terminal sequences. These processes involve a number of telomere-related proteins. A major challenge in the field is to elucidate the full constitution of telomere-associated proteins and to understand how different protein complexes are regulated at chromosome termini. Here, I report the identification and characterization of STN1 (Suppressor of cdc thirteen, 1), CTC1 (Conserved Telomere maintenance Component 1) and TEN1 (Telomeric pathways in association with Stn1, 1) in Arabidopsis. CTC1/STN1/TEN1 (CST) forms a trimeric complex that specifically associates with telomeres. Loss of any component of the CST induces catastrophic telomere loss, disrupted telomere end architecture, and massive chromosome end-to-end fusions. Thus, CST plays an essential role in chromosome end protection. I also show that CST function at telomeres is independent of a previously characterized capping complex KU70/KU80, and that ATR is responsible for a checkpoint response in plants lacking CTC1/STN1. Additionally, I present data showing that Arabidopsis POT1a (Protection Of Telomere 1, a) has evolved as a telomerase recruitment factor. Unlike POT1 in other eukaryotes which binds and protects ss telomeric DNA, AtPOT1a interacts with telomerase RNA (TER). Based on an evolutionary analysis, we found that the POT1a lineage is under positive selection in the Brassicaceae family in which Arabidopsis belongs. Mutations of two positive selection sites significantly reduce POT1a?s activity in vivo. These data suggest POT1a is under pressure to evolve from a telomeric DNA binding protein to a TER binding protein. I also discovered that POT1a interacts with the novel telomere capping protein CTC1 in vitro and in vivo. Thus, I hypothesize that POT1a acts as a telomerase recruitment factor linking this enzyme to the chromosome termini via interacting with TER and CTC1. Finally, I dissected the functional domains of POT1a and demonstrated that both the N-terminus and the C-terminus of POT1a are required for its function in vivo. In summary, my work has uncovered several new and essential telomereassociated proteins that provide new insight into mechanisms of chromosome end protection and maintenance.

Mms1 and Mms22 are important mediators of genome stability in Saccharomyces cerevisiae. Deletion mutants of both genes display decreased viability in the presence of a number of known DNA damaging agents as well as agents that perturb DNA replication. Here I present a detailed analysis of Mms1 and Mms22 function in stabilizing DNA replication forks. I find that mms1∆ and mms22∆ strains accumulate spontaneous DNA damage and have an increased rate of mutation during a normal cell cycle. Additionally, treatment with genotoxic agents causes defects in induced recombination as well as inhibiting the ability of cells to efficiently recover from DNA damage. Genetic interaction data support a model where Mms1 and Mms22 function with components of the replication fork. Accordingly I observed that the enrichment of key replication fork proteins at regions proximal to replication origins is decreased in mms1∆ and mms22∆ cells during replication stress. Furthermore, I monitored DNA replication in wild-type and mms1∆ strains and found that mms1∆ strains exhibit irregular fork progression under conditions of replication stress. I therefore concluded that Mms1 and Mms22 function at replication forks. Lastly, I uncovered that Mms1 is regulated by Rtt101-dependent ubiquitin-mediated proteolysis. Mms1 degradation is likely necessary for its function as overexpression of Mms1 results in decreased cell viability both during a normal cell cycle, and during treatment with DNA damaging agents. I found that Mms1 and Mms22 operate at sites of DNA replication to promote the stability of the genome, especially under conditions of DNA damage and replication fork stress.

In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

Normal DNA replication is blocked by DNA damage in the template strand. Translesion synthesis is a major pathway for overcoming these replication blocks. In this process, multiple non-classical DNA polymerases form a complex at the stalled replication fork called the mutasome. This complex is structurally organized by the replication accessory factor PCNA and the non-classical DNA polymerase Rev1. One of the non-classical DNA polymerases within the mutasome then catalyzes replication through the damage. Each non-classical DNA polymerase has one or more cognate lesions, which the enzyme bypasses with high accuracy and efficiency. Thus, the accuracy and efficiency of translesion synthesis depends on which non-classical DNA polymerase within the mutasome is chosen to bypass the damage. In this thesis, I discuss how the most appropriate polymerase is chosen. In so doing, I examine the components of the mutasome; the structural motifs that mediate the protein interactions in the mutasome; the methods used to study translesion synthesis; the definition of a cognate lesion; the intrinsically disordered regions that tether the polymerases to PCNA and to one another; the multiple architectures that the mutasome can adopt, such as PCNA tool belts and Rev1 bridges; and the kinetic selection model in which the most appropriate polymerase is chosen via a competition among the multiple polymerases within the mutasome. Taken together, this thesis provides and inclusive review of the current state of what is known about translesion synthesis with conclusions at its end suggesting what major questions remain and ideas of how to answer them.

Un lien étroit existe entre l’intégrité du génome et l’architecture nucléaire. Les lamines, composants majeurs de l’enveloppe nucléaire sont impliquées dans de nombreux processus nucléaires, tels que la réplication, la transcription et le maintien de l’architecture nucléaire. Il a notamment été rapporté que les lamines de type A sont impliquées dans la réparation des cassures double brin de l’ADN et la stabilité des télomères. Toutefois, peu d’études ont été réalisées sur les lamines de type B. Fait intéressant, il a été observé que l’accumulation de la lamine B1 est retrouvée dans différentes tumeurs. Cependant, les conséquences d’une dérégulation de cette lamine sur la stabilité du génome restent peu documentées.Au cours de ma thèse, je me suis intéressée à l’impact d’une dérégulation de la lamine B1 sur le maintien de la stabilité du génome, notamment sur la réparation des cassures double brin de l’ADN et la stabilité des télomères. Nous avons pu mettre en évidence que la surexpression de lamine B1 conduit à un défaut de réparation par NHEJ, associé à une diminution de recrutement de 53BP1 aux dommages radio-induits. Nous avons également démontré que la lamine B1 interagit directement avec 53BP1, protéine impliquée dans le choix de la voie de réparation, et que cette interaction est régulée en cas de dommages à l’ADN. En effet, la liaison entre ces deux protéines est rompue après dommages en condition endogène, ce qui n’est pas le cas après surexpression de la lamine B1. Ce défaut de recrutement de 53BP1 aux dommages pourrait rendre compte de la diminution de l’efficacité du NHEJ. De plus, j’ai pu identifier les domaines protéiques impliqués dans cette interaction. Il est intéressant de noter que la surexpression du domaine de la lamine B1 impliquée dans l’interaction mime la surexpression de la lamine B1 entière. Au contraire, la lamine B1 délétée de ce domaine n’a aucun impact sur le recrutement de 53BP1 et la persistance des dommages. Ces différentes données confortent notre hypothèse quant à la séquestration de 53BP1 après surexpression de lamine B1.En parallèle, nous avons pu démontrer que la surexpression de la lamine B1 entraine l’apparition de diplochromosomes concomitants à une sénescence accrue. Ce phénomène d’endoréplication peut être induit par des défauts télomériques, tels que des télomères dysfonctionnels ou déprotégés. De façon intéressante, mes données montrent que la surexpression de la lamine B1 entrainent des dommages télomériques. Nous avons également établit que la lamine B1 interagit avec TRF2, protéine du complexe « shelterin » permettant la protection des télomères contre la signalisation des dommages à l’ADN. La rétention putative de TRF2 par la lamine B1 pourrait être à l’origine des défauts télomériques observés après la surexpression de cette dernièreCette étude démontre de nouveaux rôles de la lamine B1 dans le maintien de la stabilité du génome, notamment à travers ses interactions avec deux protéines clefs dans la réparation des cassures double brin et la stabilité des télomères. Cela nous ouvre de nouvelles pistes de recherche qui permettront une meilleure compréhension des mécanismes moléculaires impliqués dans la tumorigenèse et en particulier sur le lien existant entre l’intégrité de l’architecture nucléaire et la stabilité du génome. / A close link exists between genome stability and nuclear architecture. Lamins, major component of the nuclear envelope, are involved in many nuclear processes, such as replication, transcription and nuclear architecture. It has been reported than lamins A/C are involved in double strand break repair and telomere stability. However, few studies have been conducted on B-type lamins. Interestingly, it was observed that the accumulation of lamin B1 is found in different tumors. Nevertheless, consequences of its deregulation on genome stability remain poorly documented.During my PhD, I analysed the impact of deregulation of lamin B1 on genome maintenance, including double-strand breaks repair and telomere stability. We were able to demonstrate that overexpression of lamin B1 leads to defect of NHEJ, associated with decrease of the 53BP1 recruitment to DNA damage. We have also shown that lamin B1 interacts directly with 53BP1, a protein involved in the choice of the repair pathway, and that this interaction is regulated upon DNA damage. Indeed, the association between these two proteins is disrupted after damage, in endogenous condition, in contrast this dissociation is not observed after lamin B1 overexpression. The defect of 53BP1 recruitment to DNA damage could account for the decrease in the NHEJ efficiency. Moreover, I have identify the protein domains involved in this interaction. It is interesting to note that overexpression of the interaction domain mimics the overexpression of the full lamin B1. Instead, lamin B1 deleted from this domain has no impact on 53BP1 recruitment and on DNA damage persistence. These data support our hypothesis about the sequestration 53BP1 after overexpression of lamin B1.In parallel, we have demonstrated that the lamin B1 overexpression causes the appearance of diplochromosomes concurrent to an increase of senescence. This phenomenon of endoreduplication can be induced by telomere defects such as dysfunctional or deprotected telomeres. Interestingly, I have observed that lamin B1 overexpression leads telomere damages. We also established that lamin B1 interacts with TRF2, a protein of "shelterin" complex involved in the protection against the DNA damage signaling at telomere. The putative retention TRF2 by lamin B1 could cause telomere defects observed after overexpression of the latter.This study identifies new roles of lamin B1 in maintaining genome stability, including through its interactions with two key proteins in the repair of double-strand breaks and stability of telomeres. This opens up new ways of research that will enable a better understanding of the molecular mechanisms involved in tumorigenesis and in particular on the relationship between the integrity of the nuclear architecture and genome stability.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21026号 / 医科博第87号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 篠原 隆司, 教授 松田 道行, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM