DNA is susceptible to both exogenous and endogenous damaging agents. Damage is constantly reversed by a wide range of DNA repair pathways. Lesions which escape such repair may cause nucleotide mis-pairing and stalled replication, resulting in mutagenesis and cell death, respectively if left unresolved. Stalled replication is particularly dangerous because replication fork collapse can lead to double-strand breaks (DSBs) and chromosome rearrangement, a hallmark of cancer. DNA damage tolerance (DDT) is defined as a mechanism that allows DNA synthesis to occur in the presence of replication-blocking lesions.<p> DDT, also known as post-replication repair (PRR) in yeast, has been well characterized in the lower eukaryotic model Saccharomyces cerevisiae to consist of error-free and error-prone (mutagenic) pathways. Mono-ubiquitination of proliferating cell nuclear antigen (PCNA) by the Rad6-Rad18 complex promotes mutagenesis by recruiting low fidelity translesion synthesis (TLS) polymerases, while continual Lys63-linked poly-ubiquitination of PCNA by the Mms2-Ubc13-Rad5 complex promotes error-free lesion bypass. Since most of the genes involved in DNA metabolism are conserved within eukaryotes, from yeast to human, I tested the hypothesis that mammalian cells also possess two-pathway DDT in response to DNA damage. Namely, the error-free pathway is dependent on the Ubc13-Mms2 complex, while the error-prone pathway utilizes the TLS polymerases, such as Rev3.<p> By utilizing cultured mammalain cells and producing antibodies against human Ubc13, Mms2 and Rev3, I was able to show that all three proteins associate with PCNA in S-phase cells, and that this association is enhanced following DNA damage. Ubc13-Mms2 association with PCNA was enhanced in response to DSBs. Furthermore, suppression of Ubc13 or Mms2 using interfering RNA technology resulted in increased spontaneous DSBs. In response to UV exposure, Rev3 co-localized with PCNA and two other TLS polymerases, Rev1 and Pol-Ø, at the damage site. UV-induced Rev3 nuclear focus formation was dependent on Rev1 but independent of Pol-£b. Surprisingly, over-expression of Pol-£b was sufficient to induce spontaneous Rev3 nuclear foci. It was further demonstrated that Rev1 and Pol-Ø were independently recruited to the damage site and did not require Rev3. These observations support and extend the polymerase switch model which regulates the activity of the replicative and TLS polymerases. Finally, simultaneous suppression of Rev3 along with Ubc13 or Mms2 resulted in a synergistic sensitivity to UV, whereas simultaneous suppression of Ubc13 and Pol-Ø resulted in an additive effect. These results are consistent with those in yeast cells, implying a comparable mammalian two-pathway DDT model.<p> Additional interesting observations were made. Firstly, Ubc13 interacts with Uev1A, a close homolog of Mms2, which is involved in the NF-£eB signaling pathway independent of DNA damage. Secondly, Rev3 appears to be excluded from the nucleus in a fraction of low passage normal non-S-phase cells, whereas in tumor derived cell lines, Rev3 is consistently enriched in the nucleus independent of cell cycle stage. Finally, Rev3 is elevated during mitosis and associates with condensed chromosomes, suggesting a possible novel role in mitosis. Consistent with this notion, chronic ablation of Rev3 resulted in cell death with inappropriate chromosome segregations. The above preliminary observations require further investigation.

DNA is susceptible to both exogenous and endogenous damaging agents. Damage is constantly reversed by a wide range of DNA repair pathways. Lesions which escape such repair may cause nucleotide mis-pairing and stalled replication, resulting in mutagenesis and cell death, respectively if left unresolved. Stalled replication is particularly dangerous because replication fork collapse can lead to double-strand breaks (DSBs) and chromosome rearrangement, a hallmark of cancer. DNA damage tolerance (DDT) is defined as a mechanism that allows DNA synthesis to occur in the presence of replication-blocking lesions.<p> DDT, also known as post-replication repair (PRR) in yeast, has been well characterized in the lower eukaryotic model Saccharomyces cerevisiae to consist of error-free and error-prone (mutagenic) pathways. Mono-ubiquitination of proliferating cell nuclear antigen (PCNA) by the Rad6-Rad18 complex promotes mutagenesis by recruiting low fidelity translesion synthesis (TLS) polymerases, while continual Lys63-linked poly-ubiquitination of PCNA by the Mms2-Ubc13-Rad5 complex promotes error-free lesion bypass. Since most of the genes involved in DNA metabolism are conserved within eukaryotes, from yeast to human, I tested the hypothesis that mammalian cells also possess two-pathway DDT in response to DNA damage. Namely, the error-free pathway is dependent on the Ubc13-Mms2 complex, while the error-prone pathway utilizes the TLS polymerases, such as Rev3.<p> By utilizing cultured mammalain cells and producing antibodies against human Ubc13, Mms2 and Rev3, I was able to show that all three proteins associate with PCNA in S-phase cells, and that this association is enhanced following DNA damage. Ubc13-Mms2 association with PCNA was enhanced in response to DSBs. Furthermore, suppression of Ubc13 or Mms2 using interfering RNA technology resulted in increased spontaneous DSBs. In response to UV exposure, Rev3 co-localized with PCNA and two other TLS polymerases, Rev1 and Pol-Ø, at the damage site. UV-induced Rev3 nuclear focus formation was dependent on Rev1 but independent of Pol-£b. Surprisingly, over-expression of Pol-£b was sufficient to induce spontaneous Rev3 nuclear foci. It was further demonstrated that Rev1 and Pol-Ø were independently recruited to the damage site and did not require Rev3. These observations support and extend the polymerase switch model which regulates the activity of the replicative and TLS polymerases. Finally, simultaneous suppression of Rev3 along with Ubc13 or Mms2 resulted in a synergistic sensitivity to UV, whereas simultaneous suppression of Ubc13 and Pol-Ø resulted in an additive effect. These results are consistent with those in yeast cells, implying a comparable mammalian two-pathway DDT model.<p> Additional interesting observations were made. Firstly, Ubc13 interacts with Uev1A, a close homolog of Mms2, which is involved in the NF-£eB signaling pathway independent of DNA damage. Secondly, Rev3 appears to be excluded from the nucleus in a fraction of low passage normal non-S-phase cells, whereas in tumor derived cell lines, Rev3 is consistently enriched in the nucleus independent of cell cycle stage. Finally, Rev3 is elevated during mitosis and associates with condensed chromosomes, suggesting a possible novel role in mitosis. Consistent with this notion, chronic ablation of Rev3 resulted in cell death with inappropriate chromosome segregations. The above preliminary observations require further investigation.

DNA damage tolerance (DDT) is a process utilized by cells to bypass replication blocking lesions in the DNA, preventing replication fork collapse and maintaining genomic stability and cell viability. In Saccharomyces cerevisiae DDT consists of two branched pathways. One branch allows direct replication past lesions in the DNA utilizing specific error-prone polymerases, a process known as translesion DNA synthesis (TLS). The other branch utilizes homologous recombination and template switch to replicate past damaged DNA in an error-free manner. RAD5 has traditionally been characterized as belonging to the error-free pathway of DNA damage tolerance. The protein is multi-functional, with several specific activities identified and classified to the error-free branch of DDT. However, there is also evidence for additional uncharacterized activities of the protein. The goal of this research was to determine which branches of DNA damage tolerance the uncharacterized activities of Rad5 are involved in. A two-pronged approach was utilized, elucidation of the physical interactions of the protein, and examination of the genetic interactions between RAD5 and other DDT genes. The evidence indicates that Rad5 plays a partial role in TLS and the protein is known to physically interact with Rev1, a member of the TLS pathway. We assumed this physical interaction mediates the TLS activity of Rad5. The yeast two-hybrid assay was utilized to examine the interaction between Rev1 and truncated Rad5 fragments, and the N-terminal 30 amino acids of Rad5 proved sufficient to maintain the interaction. This research sets the stage to identify key residues in Rad5 for the interaction with Rev1, and the creation of a TLS deficient rad5 mutant by targeting those key residues. Genetic interactions between RAD5 and genes required for the initiation of DDT in the cell were examined based on sensitivity to killing by various DNA damaging agents. We determined that the functions of Rad5 rely on PCNA modification, and thus do not function in a cellular process unrelated to Rad5. Potential uncharacterized functions are discussed on the basis of these results and the results of the interaction studies. Future structural and functional studies are proposed to better understand the role of Rad5 in the cell.

DNA-damage tolerance (DDT) in yeast is composed of two parallel pathways and mediated by sequential ubiquitination of proliferating cell nuclear antigen (PCNA). While monoubiquitination of PCNA promotes translesion synthesis (TLS), which is dependent on low fidelity polymerase ζ (Pol ζ) composed of a catalytic subunit Rev3 and a regulatory subunit Rev7, polyubiquitination of PCNA by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Inactivation of these two pathways results in a synergistic effect on DNA-damage responses; however, this two-branch DDT model has not been reported in any multicellular organisms. In order to examine whether Arabidopsis thaliana possesses a two-branch DDT system, rad5a rev3 double mutant plants were created and compared with the corresponding single mutants. Arabidopsis rad5a and rev3 mutations are indeed synergistic with respect to growth inhibition induced by replication-blocking lesions, suggesting that AtRAD5a and AtREV3 are required for error-free and TLS branches of DDT, respectively. Unexpectedly this study reveals three modes of genetic interactions in response to different types of DNA damage, indicating that plant RAD5 and REV3are also involved in DNA damage responses independent of DDT. By comparing with yeast cells, it is apparent that plant TLS is a more frequently utilized means of lesion bypass than error-free DDT. In addition, it was also observed that treatments with the DNA damaging agent methylmethanesulfonate increased the nuclear ploidy level in the double mutant plants.

Notre génome subit constamment les effets néfastes des agents endommageant de l'ADN. Afin de se protéger de ces effets délétères, les cellules disposent d’un système de détection des dommages à l’ADN (point de contrôle ou « checkpoint »). Certaines lésions peuvent persister quand les cellules entrent en phase S et inhiber ainsi la synthèse de l’ADN en interférant avec les ADN polymérases réplicatives. Ceci peut provoquer des arrêts prolongés des fourches de réplication ce qui fragilise l’ADN. Pour préserver l’intégrité de l’information génétique, les cellules ont développé une voie de tolérance qui implique des ADN polymérases spécialisées dans la réplication des lésions, appelées ADN Polymérases translésionnelles (Pols TLS). Dans ce processus, PCNA joue le rôle de facteur d’échafaudage pour de nombreuses protéines impliquées dans le métabolisme de l'ADN. Les mécanismes de régulation des échanges entre les différents partenaires de PCNA ne sont pas très bien compris. Parmi les protéines qui interagissent avec PCNA, CDT1, p21 ou encore PR-Set7/Set8 sont caractérisées par une forte affinité pour cette protéine. Ces dernières possèdent un motif d’interaction particulier avec PCNA, nommé « PIP degron », qui favorise leur protéolyse d'une manière dépendante de l’E3 ubiquitine ligase CRL4Cdt2. Après irradiation aux UV-C, le facteur d’initiation de la réplication CDT1 est rapidement détruit d’une manière dépendante de son PIP degron, Dans la première partie de mon travail, j’ai contribué à comprendre le rôle fonctionnel de cette dégradation. Les résultats obtenus ont fourni des évidences expérimentales qui montrent que l’inhibition de la dégradation de CDT1 par CRL4Cdt2 dans les cellules de mammifères compromet la relocalisation des TLS Pol eta et Pol kappaen foyers nucléaires induits par les irradiations UV-C. On a constaté que seules les protéines qui contiennent un PIP degron interfèrent avec la formation de foyers de Pol eta. La mutagenèse du PIP degron de CDT1 a révélé qu'un résidu de thréonine conservé parmi les PIP degrons est essentiel pour l'inhibition de la formation des foyers des TLS Polymérases. Les résultats obtenus suggèrent que l’élimination de protéines contenant des PIP degrons par la voie CRL4Cdt2 régule le recrutement de TLS Polymérases au niveau des sites des dommages induits par les UV-C.Dans un second temps, on s’est intéressé à l’étude du checkpoint des dommages à l’ADN au cours de l’embryogénèse. En effet, dans les embryons précoces, le checkpoint est silencieux jusqu'à la transition de mid-blastula (MBT), en raison de facteurs maternels limitants. Dans ce travail, nous avons montré, aussi bien in vitro qu’in vivo, que l’ubiquitine ligase de type E3 RAD18, un régulateur majeur de la translésion, est un facteur limitant pour l’activation du checkpoint dans les embryons de xénope. Nous avons montré que l'inactivation de la fonction de l’ubiquitine ligase RAD18 conduit à l'activation du checkpoint par un mécanisme qui implique l’arrêt des fourches de réplication en face des lésions produites par les UV-C. De plus, nous avons montré que l'abondance de RAD18 et de PCNA monoubiquitiné (PCNAmUb) est régulée au cours de l’embryogénèse. À l’approche de la MBT, l’abondance de l'ADN limite la disponibilité de RAD18, réduisant ainsi la quantité de PCNAmUb et induisant la dé-répression du checkpoint. En outre, nous avons montré que cette régulation embryonnaire peut être réactivée dans les cellules somatiques de mammifères par l'expression ectopique de RAD18, conférant une résistance aux agents qui causent des dommages à l'ADN. Enfin, nous avons trouvé que l'expression de RAD18 est élevée dans les cellules souches cancéreuses de glioblastome hautement résistantes aux dommages de l'ADN. En somme, ces données proposent RAD18 comme un facteur embryonnaire critique qui inhibe le point de contrôle des dommages de l’ADN et suggèrent que le dérèglement de l’expression de RAD18 peut avoir un potentiel oncogénique inattendu / Our genome is continuously exposed to DNA damaging agents. In order to preserve the integrity of their genome, cells have evolved a DNA damage signalling pathway known as checkpoint. Some lesions may persist when cells enter the S-phase and halt the progression of replicative DNA polymerases. This can cause prolonged replication forks stalling which threaten the stability of the genome. To preserve the integrity of genetic information, cells have developed a tolerance pathway which involves specialized DNA polymerases, called translesion DNA polymerases (TLS Pols). These polymerases have the unique ability to accommodate the damaged bases thanks to their catalytic site. In this process, PCNA acts as a scaffold for many proteins involved in DNA metabolism. The mechanisms governing the exchanges between different PCNA partners are not well understood. Among the proteins that interact with PCNA, CDT1, p21 and PR-Set7/set8 are characterized by a high binding affinity. These proteins have a particular interaction domain with PCNA, called "PIP degron", which promotes their proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. After UV-C irradiation, the replication initiation factor CDT1 is rapidly degraded in a PIP degron-dependent manner. During the first part of my work, we wanted to understand the functional role of this degradation. Our results have shown that inhibition of CDT1 degradation by CRL4Cdt2 in mammalian cells, compromises the relocalisation of TLS Pol eta and Pol kappato nuclear foci after UV-C irradiation. We also found that only the proteins which contain a PIP degron interfere with the formation of Pol eta foci. Mutagenesis experiments on CDT1 PIP degron revealed that a threonine residue conserved among PIP degrons is essential for inhibiting foci formation of at least two TLS polymerases. This results suggest that CRL4Cdt2-dependent degradation of proteins containing PIP degrons regulates the recruitment of TLS polymerases at sites of UV-induced DNA damage.During the second part of my thesis, we studied DNA damage checkpoint regulation during embryogenesis. Indeed, in early embryos, the DNA damage checkpoint is silent until the mid-blastula transition (MBT) due to maternal inhibiting factors. In this work, we have shown, both in vitro and in vivo, that the E3 ubiquitin ligase RAD18, a major regulator of translesion DNA synthesis, is a limiting factor for the checkpoint activation in Xenopus embryos. We have also shown that RAD18 depletion leads to the activation of DNA damage checkpoints by inducing replication fork uncoupling in front of the lesions. Furthermore, we showed that the abundance of RAD18 and PCNA monoubiquitination (PCNAmUb) is regulated during embryonic development. Near the MBT, the increased abundance of DNA limits the availability of RAD18, thereby reducing the amount of PCNAmUb and inducing the de-repression of the checkpoint. Moreover, we have shown that this embryonic-like regulation can be reactivated in somatic mammalian cells by ectopic expression of RAD18, conferring resistance to DNA damaging. Finally, we found high RAD18 levels in glioblastoma cancer stem cells highly resistant to DNA damage. All together, these data propose RAD18 as a critical factor that inhibits DNA damage checkpoint in early embryos and suggests that dysregulation of RAD18 expression may have an unexpected oncogenic potential

Ubiquitination is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitination requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets proteins for degradation, while the noncanonical Lys63-linked polyubiquitination of the proliferating cell nuclear antigen is required for error-free DNA damage tolerance (DDT or postreplication repair) in yeast. The ubiquitin-conjugating enzyme <i>Ubc13</i> and a cognate Ubc enzyme variant (Uev or Mms2) are involved in this process. Because there is less information available on either Lys63-linked ubiquitination or error-free DDT in plants, the goal of my research was to study the functions of <i>Ubc13</i> and Uev in plants using <i>Arabidopsis thaliana</i> as the model organism.<p> Four <i>UEV1</i> genes from <i>Arabidopsis thaliana</i> were isolated and characterized. All four <i>Uev1</i> proteins can form a stable complex with AtUbc13 and can promote <i>Ubc13</i> mediated Lys63 polyubiquitination. All four <i>UEV1</i> genes can replace yeast MMS2 in DDT function in vivo. Although these genes are ubiquitously expressed in most tissues, <i>UEV1D</i> appears to be expressed at a much higher level in germinating seeds and pollen. We obtained and characterized two <i>uev1d</i> null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Seeds that germinated grew slow and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage agent. These results indicate that <i>Ubc13-Uev</i> complex functions in DNA damage response in <i>Arabidopsis thaliana.</i> <i>Arabidopsis thaliana</i> contains two <i>UBC13</i> genes, AtUBC13A and AtUBC13B, that are highly conserved with respect to DNA sequence, protein sequence and genomic organization, suggesting that they are derived from a recent gene duplication event. Both <i>AtUbc13</i> proteins are able to physically interact with human and yeast Mms2, implying that plants also employ a Lys63-linked polyubiquitination reaction. Furthermore, Both <i>AtUBC13</i> genes were able to functionally complement the yeast ubc13 null mutants, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The <i>AtUBC13</i> genes appear to be expressed ubiquitously and were not induced by various conditions tested.<p> The <i>ubc13a/b</i> double mutant lines were created and displayed strong phenotypic changes. The double mutant plants were delayed in seed germination as well as cotyledon and true leaf development. When seedlings were grown vertically on plates, the roots of the double mutant were shorter and grew in a zig-zag manner, compared to the straight growth of wild type roots. Root length and number of lateral roots on wild type and <i>ubc13a</i> and <i>ubc13b</i> single mutant plants were about 3 times longer than those of double mutant plants after 9 and 12 days of growth. When double mutant seeds were sown directly into soil, many did not germinate and those that germinated grew much slower than wild type. At 35 days, double mutant plants were smaller with thinner, flatter, and lighter coloured rosette leaves compared to wild type plants. These phenotypes indicate that <i>AtUbc13</i> not only plays a role in DDT to protect genome integrity but also is involved in plant development. Hence, this study set a cornerstone for future investigations into the roles of <i>Ubc13</i> and <i>Uev1</i> in plant development.

Ubiquitination is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitination requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets proteins for degradation, while the noncanonical Lys63-linked polyubiquitination of the proliferating cell nuclear antigen is required for error-free DNA damage tolerance (DDT or postreplication repair) in yeast. The ubiquitin-conjugating enzyme <i>Ubc13</i> and a cognate Ubc enzyme variant (Uev or Mms2) are involved in this process. Because there is less information available on either Lys63-linked ubiquitination or error-free DDT in plants, the goal of my research was to study the functions of <i>Ubc13</i> and Uev in plants using <i>Arabidopsis thaliana</i> as the model organism.<p> Four <i>UEV1</i> genes from <i>Arabidopsis thaliana</i> were isolated and characterized. All four <i>Uev1</i> proteins can form a stable complex with AtUbc13 and can promote <i>Ubc13</i> mediated Lys63 polyubiquitination. All four <i>UEV1</i> genes can replace yeast MMS2 in DDT function in vivo. Although these genes are ubiquitously expressed in most tissues, <i>UEV1D</i> appears to be expressed at a much higher level in germinating seeds and pollen. We obtained and characterized two <i>uev1d</i> null mutant T-DNA insertion lines. Compared with wild-type plants, seeds from uev1d null plants germinated poorly when treated with a DNA-damaging agent. Seeds that germinated grew slow and the majority ceased growth within 2 weeks. Pollen from uev1d plants also displayed a moderate but significant decrease in germination in the presence of DNA damage agent. These results indicate that <i>Ubc13-Uev</i> complex functions in DNA damage response in <i>Arabidopsis thaliana.</i> <i>Arabidopsis thaliana</i> contains two <i>UBC13</i> genes, AtUBC13A and AtUBC13B, that are highly conserved with respect to DNA sequence, protein sequence and genomic organization, suggesting that they are derived from a recent gene duplication event. Both <i>AtUbc13</i> proteins are able to physically interact with human and yeast Mms2, implying that plants also employ a Lys63-linked polyubiquitination reaction. Furthermore, Both <i>AtUBC13</i> genes were able to functionally complement the yeast ubc13 null mutants, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The <i>AtUBC13</i> genes appear to be expressed ubiquitously and were not induced by various conditions tested.<p> The <i>ubc13a/b</i> double mutant lines were created and displayed strong phenotypic changes. The double mutant plants were delayed in seed germination as well as cotyledon and true leaf development. When seedlings were grown vertically on plates, the roots of the double mutant were shorter and grew in a zig-zag manner, compared to the straight growth of wild type roots. Root length and number of lateral roots on wild type and <i>ubc13a</i> and <i>ubc13b</i> single mutant plants were about 3 times longer than those of double mutant plants after 9 and 12 days of growth. When double mutant seeds were sown directly into soil, many did not germinate and those that germinated grew much slower than wild type. At 35 days, double mutant plants were smaller with thinner, flatter, and lighter coloured rosette leaves compared to wild type plants. These phenotypes indicate that <i>AtUbc13</i> not only plays a role in DDT to protect genome integrity but also is involved in plant development. Hence, this study set a cornerstone for future investigations into the roles of <i>Ubc13</i> and <i>Uev1</i> in plant development.

Faithful replication of genomic DNA is crucial for the survival of a cell. In order to achieve high-level accuracy in copying its genome, all cells employ replicative DNA polymerases that have intrinsic high fidelity. When an error occurs on the template DNA strand, in the form of lesions caused by diverse chemicals, reactive oxygen species, or UV light, the high-fidelity replicative DNA polymerases are stalled. To bypass these replication blocks, cells harbor multiple specialized translesion DNA polymerases that are error-prone and therefore able to accommodate the lesions and continue DNA synthesis. As a result of their low fidelity, the translesion polymerases are associated with increased mutagenesis, drug resistance, and cancer. Therefore, the access of the translesion polymerases to DNA needs to be tightly controlled, but how this is achieved has been the subject of debate. This Thesis presents the development of a co-localization single-molecule spectroscopy (CoSMoS) method to directly visualize the loading of the Escherichia coli replicative polymerase on DNA, as well as the exchange between the replicative polymerase and the translesion polymerases Pol II and Pol IV. In contrast to the toolbelt model for the exchange between the polymerases, this work shows that the translesion polymerases Pol II and Pol IV do not form a stable complex with the replicative polymerase Pol IIIα on the β-clamp. Furthermore, we find that the sequential activities of the replication proteins: clamp loader, clamp, and Pol IIIα, are highly organized while the exchange with the translesion polymerases is disordered. This exchange is not determined by lesion-recognition but instead a concentration-dependent competition between the replicative and translesion polymerases for the hydrophobic groove on the surface of the β-clamp. Hence, our results provide a unique insight into the temporal organization of events in DNA replication and translesion synthesis.

No description available.