Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are non-enveloped, circular double-stranded DNA viruses that infect basal keratinocytes of stratified squamous epithelia. High-risk HPV (HR-HPV) infection causes nearly all cervical cancers and an increasing number of head and neck cancers. While prophylactic vaccinations have reduced the incidence of HPV infection and attributable cancers, currently there is no cure for pre-existing HPV infection. As such, HPV remains a global health threat and a better understanding of HPV biology remains of significant medical importance for identification of novel therapeutic targets. The multi-subunit structural maintenance of chromosomes 5/6 complex (SMC5/6) is comprised of SMC5, SMC6 and NSE1-4. SMC5/6 is essential for homologous recombination DNA repair and reportedly functions as an antiviral factor during hepatitis B and herpes simplex-1 viral infections. Intriguingly, SMC5/6 has been found to associate with HR-HPV E2 proteins, which are multifunctional transcription factors essential to regulation of viral replication and transcription. The function of SMC5/6 associations with E2, as well as its role during HR-HPV infection remain unclear and we explored this question in the context of HR-HPV- 31. SMC6 interacted with HPV-31 E2 and co-immunoprecipitation of SMC6/E2 complexes required the E2 transactivation domain, inferring SMC6 association is limited to the full-length E2 isoform. Depletion of SMC6 and NSE3 increased HPV replication and transcription in keratinocytes stably maintaining episomal HPV-31, suggesting that the SMC5/6 complex represses these processes. Neither SMC6 nor NSE3 co-IP the viral E1 DNA helicase alone or E1/E2 complexes but the association of SMC6 with E2 was reduced in the presence of E1, indicating that SMC6 competes with E1 for E2 binding. This infers that SMC6 repression of the viral replicative program may involve inhibiting initiation of viral replication by disrupting E2 interactions with E1. Chromatin immunoprecipitation determined that SMC6 is present on episomal HPV-31 genomes, alluding to a possible role for SMC5/6 in modifying the chromatin state of viral DNA. Taken together, these findings describe a novel function for SMC5/6 as a repressor of the HPV-31 replicative program.

Les télomères sont des structures nucléoprotéiques formées de séquences d’ADN répétées associées à des protéines spécialisées assurant la protection des extrémités des chromosomes eucaryotes et leur réplication complète. La télomérase est une ribonucléoprotéine catalysant l’ajout de répétitions télomérique pour contrer la perte de séquences inhérente à la réplication des extrémités des chromosomes linéaires. Plusieurs facteurs jouent des rôles importants dans le maintien de l’intégrité des télomères. Le champ d’étude de la biologie des télomères est toujours en expansion, étant donné les liens étroits entre les télomères, le cancer et le vieillissement. Les travaux présentés dans cette Thèse se divisent en trois parties. Les deux premières utilisent la levure Saccharomyces cerevisiae pour explorer les liens entre le complexe Smc5/6 et le maintien des télomères, ainsi que les mécanismes de terminaison de transcription de la composante ARN de la télomérase (T1c1). La troisième partie est une étude épidémiologique préliminaire examinant la taille des télomères de patients atteints de polyarthrite rhumatoïde (PR). Premièrement, les protéines SMC (structural maintenance of chromosomes) forment 3 complexes conservés requis pour la transmission des chromosomes lors des divisions cellulaires. Le complexe Smc5/6 a été impliqué dans la réparation de l’ADN et le maintien des télomères. Les rôles des complexes SMC, en particulier Smc5/6, dans la biologie des télomères ont donc été investigués. Les résultats montrent que les complexes SMC sont importants pour la survie pendant la sénescence en absence de télomérase. Les données obtenues pour Smc5/6 supportent un modèle dans lequel le complexe est requis pour la réplication complète et la réparation adéquate des chromosomes. De fréquentes cassures d’ADN au niveau des répétitions télomériques sont observées en absence du complexe, illustrant l’importance particulière de Smc5/6 pour la séparation des régions télomériques. Deuxièmement, il est établi que l’abondance de la télomérase est critique pour l’homéostasie des télomères. Mais le contrôle de l’expression de l’ARN T1c1 est encore obscur. L’analyse des séquences en 3' du gène TLC1 révèle des signaux reconnus par les 2 voies de terminaison de transcription par l’ARN polymérase II. Les résultats indiquent que la formation de T1c1 est contrôlée par la voie de terminaison des petits ARNs noncodants Nrd1/Nab3. T1c1 existe sous 2 formes, une majeure, non polyadénylée, présente dans la télomérase active, et une mineure, polyadénylée, dont le rôle est inconnu. Les données montrent que la synThèse et la fonction de l’ARN T1c1 mature ne nécessitent pas la forme polyA+ comme précurseur. La terminaison dépendante de la polyadénylation pourrait être un mécanisme de sûreté pour arrêter la transcription. Troisièmement, plusieurs maladies sont associées à des défauts dans le maintien des télomères. La PR est une maladie auto-immune causant une inflammation chronique et une destruction graduelle des articulations. Des études suggèrent que les lymphocytes de patients atteints de PR auraient des télomères anormalement courts. Ensuite, des marqueurs pronostiques fiables identifiant les patients qui développeront une arthrite persistante et sévère font actuellement défaut, mais seraient utiles afin d’élaborer des traitements plus efficaces. Une étude épidémiologique a donc été amorcée pour analyser l’érosion des télomères des lymphocytes dans une cohorte de patients avec arthrite débutante et évaluer de potentielles corrélations avec la progression et la sévérité de la maladie. Les résultats préliminaires obtenus par 3 techniques de mesure de la taille des télomères (TRF, STELA et qPCR) sont incomplets, mais semblent indiquer un défaut dans le maintien des télomères chez les patients. Les observations illustrent aussi les limites des études épidémiologiques longitudinales analysant la taille des télomères.

No description available.

L’infection par le virus de l’hépatite B (HBV) est problème majeur de santé publique mondial car, en dépit d’un vaccin efficace, les traitements curatifs actuels ne permettent pas l’élimination complète du virus. Comprendre les mécanismes de réplication du virus et son rôle dans la survenue du cancer hépatocellulaire (CHC) reste un enjeu majeur.Le rôle de la protéine HBx dans l’infection par HBV et l’oncogenèse viro-induite, reste mal connu, malgré un grand nombre de publications, car les fonctions décrites jusqu'alors sont limitées à des contextes d’études particuliers, souvent loin des conditions physiologiques.Mes travaux de thèse reposent sur l’utilisation de modèles d’études proches de la physiologie naturelle d’une infection par HBV, notamment des cellules primaires infectables in vitro. J’ai pu démontrer lors de mon étude que HBx est indispensable à la réplication de HBV, et qu’il agit essentiellement via son interaction avec DDB1 pour contrer la restriction du virus due au complexe SMC5/6, en induisant sa dégradation. Ce facteur de restriction permet de bloquer la transcription de l’ADN viral au niveau épigénétique. Ce nouveau rôle inattendu de SMC5/6 ouvre de nombreux axes de recherche, notamment sur les mécanismes de restriction des virus à ADN épisomal. SMC5/6 est connu pour son implication dans les voies de réparation de l’ADN : la dernière partie de ce manuscrit montre que sa dégradation dans les cellules infectées, altère ces mécanismes et sensibilise les cellules aux dommages à l’ADN, induits notamment par la radiothérapie. La présence de HBx dans les CHC pourrait ainsi s’avérer un atout pour le traitement du CHC / Hepatitis B virus (HBV) infection is a major health problem worldwide as (1) despite an effective preventive vaccine over 240 million individuals are chronically infected and (2) the actual viral suppressive treatments available do not eliminate viral DNA from cells. Thus, infected individuals are at a high risk of developing hepatocellular carcinoma (HCC) and understanding viral replication mechanisms and how it impacts on hepatocarcinogenesis is a major challenge.The role of the HBx protein, notably in viral replication and oncogenic processes, is the subject of many publications. However, many studies have often used non-physiological infection conditions. My thesis project has addressed these limitations by using cellular models, including primary human hepatocytes which can be infected by HBV, to investigate HBx’s role in these processes. I have shown that HBx is indispensable for HBV replication and that HBx associates with the infected cell’s DDB1/ E3 ubiquitin complex to target its Smc5/6 complex for degradation via the proteasome. These studies have identified that the Smc5/6 complex is a novel viral restriction factor that acts at an epigenetic level to block viral replication. This unexpected role of SMC5/6 has led to new research into the evolutionary conservation of restriction factors for episomal DNA viruses. As SMC5/6 is implicated in DNA Damage Repair (DDR), the last section of my thesis reports how SMC5/6 degradation in infected cells can sensitise cells to the cell killing effects of DNA damaging agents such as ionizing radiation and hydroxyurea. These results open-up possibilities for HCC treatment where HBx expression may be of therapeutic benefit

No description available.

DNA repair pathways have evolved to protect the genome from damage caused by intrinsic and extrinsic factors. Although numerous DNA repair mechanisms have been studied and reported, information regarding how they coordinate with the necessary changes in chromatin structure is scarce. Smc (structural maintenance of chromosomes) proteins are a conserved, essential family of proteins required for chromosome organization and accurate segregation. The budding yeast, Saccharomyces cerevisiae has three Smc-protein complexes: Smc1/3 complex (cohesin), Smc2/4 complex (condensin) and the Smc5/6 complex, required for sister chromatid cohesion, condensation and DNA repair, respectively. The chromatin associated Smc5/6 complex consists of Smc5, Smc6 and six non-smc elements (Nse1-Nse6). Smc5 and Smc6 are required for stability of repetitive chromosomal regions and sister chromatid recombination-mediated repair of double-strand breaks. Mms21/Nse2, a subunit of the Smc5/6 complex, is a SUMO E3-ligase, which conjugates SUMO (small ubiquitin-like modifier) to Smc5 and Yku70 (DNA repair protein) and its SUMO ligase activity protects the cells from extrinsic DNA damage. To address the role of Nse2 SUMO ligase in cellular events, we isolated mutants (nse2∆sl and nse2C221A) defective in the E3-ligase domain of Nse2 and found that these mutants are sensitive to genotoxic agents, for example MMS, UV or bleomycin, as expected. We found that cysteine 221 present in the SP-RING domain of Nse2 is required in the function of Nse2 in resisting genotoxic stress. We found that nse2∆sl cultures are slow growing and show increased abundance of cells having 2N DNA content (indicative of a G2-M cell cycle delay or arrest) relative to wild type cells. The DNA damage checkpoint pathway is activated to a limited extent in unchallenged nse2∆sl mutant cells indicating that cells lacking the SUMO ligase activity of Nse2 incur spontaneous DNA damage. Furthermore nse2∆sl cells are exquisitely sensitive to caffeine, an agent known to override the DNA damage checkpoint in a number of organisms by inhibiting the DNA damage checkpoint transducer ATR (Homo sapiens), Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe). In order to investigate the importance of the DNA damage checkpoint pathway for nse2∆sl cells, we employed a genetic approach. We found that nse2∆sl exhibits synthetic sick interaction with mec1∆ but not tel1∆ (defective in Mec1 or Tel1 PI kinases) or mrc1∆ (defective in Mrc1 or mediator of replication checkpoint 1) indicating that the DNA damage induced Mec1 dependent checkpoint pathway is selectively required but the replication stress checkpoint pathway is dispensable for optimal growth of unchallenged nse2∆sl cells. In order to further investigate the role of Nse2 in S phase events, we used camptothecin (CPT), a drug that induces S phase specific double strand breaks. CPT inhibits topoisomerase I by trapping the covalent Top1-DNA intermediate. Collision of a DNA replication fork with such a complex results in double-strand and single-strand breaks in DNA. We found that nse2∆sl is CPT-sensitive and that nse2∆sl top1-8 has a synthetic sick phenotype. Thus, our chemical and genetic interaction studies suggest that the SUMO ligase activity of Nse2 may be required when Top1 function is compromised. Interestingly, human and yeast Top1 proteins are known to be sumoylated. Our findings suggest that MMS-induced enhancement of Top1 sumoylation in budding yeast is partially dependent on SUMO ligase activity of Nse2. Since both sumoylation and Top1 play a role in telomere maintenance, we also examined the telomere length in single as well as double mutants and found that there is slight telomere lengthening in nse2∆sl top1-8 double mutant. To gain further insight into the genetic interaction between Nse2 and other proteins which affect DNA topology, we also investigated genetic interaction of Nse2 with other topoisomerases. We found that top3-2 nse2∆sl exhibited a synthetic sick phenotype but nse2∆sl top2-4 showed partial rescue of temperature sensitivity. In order to investigate whether chromosome integrity is compromised in nse2∆sl cells we employed a YAC (yeast artificial chromosome) based assay to examine GCRs (gross chromosomal rearrangements). We found elevated levels of GCR in nse2∆sl cells compared to wild type cells. Furthermore, deletion of DNA Topoisomerase1 in nse2∆sl background selectively destabilizes a longer YAC relative to shorter YACs. We also examined the effect of varying origin number on YAC stability in nse2∆sl as well as top1∆ and nse2∆sl top1∆ cells. We found that a YAC having fewer origins is not destabilized in nse2∆sl and top1∆ single mutants but is destabilized in the nse2∆sl top1∆ double mutant. Since Nse2 is a non-SMC member of the Smc5/6 complex, we also investigated the effect of varying origin number on YAC stability in smc6-56 and smc656 top1∆ mutants. We found that the stability of a YAC is modestly compromised in the smc6-56 mutant but its derivative having fewer origins is not further destabilized, rather it seems to be stabilized. In order to gain molecular insights into the involvement of the SUMO ligase activity of Nse2 in maintenance of chromosome integrity, we examined sumoylation of specific substrates following a candidate approach. Smc5 and Yku70 are known targets of Nse2dependent sumoylation. We found that Smc6 is also sumoylated and that the MMS-induced enhancement of Smc6 sumoylation in budding yeast is partially dependent on Nse2. To understand the functional significance of Smc5 sumoylation, we mutated lysine residues of all the four predicted sumoylation sites ψKXE/D, individually as well as all four together. We found that all the single as well as quadruple mutants were weakly sensitive to MMS suggesting that these putative sumoylation sites of Smc5 may contribute towards countering MMS-induced DNA damage. Interestingly, we found that Smc5 sumoylation is enhanced when treated with MMS (methyl methane sulfonate) but not significantly with HU (hydroxyurea) and CPT (camptothecin). We also generated putative ATP-binding defective mutants in Smc5. Previous studies suggest that the ATPase motif is required for the essential function of some Smc proteins (for example, Smc1 and Smc6). We found that smc5K75E and smc5K75Q, having a mutation in the lysine residue of the conserved GXGKS motif present in the Walker A type box at the Nterminus exhibited a null phenotype implying that this conserved lysine residue is required for essential function of Smc5. In this study, employing genetic and biochemical methods, we have characterized the Nse2 SUMO ligase defective mutant and analyzed its role in the unperturbed mitotic cell cycle and in genome maintenance. We have also employed genetic methods to study the involvement of both Nse2 and DNA Topoisomerase I in maintaining genomic stability. Lastly, we have addressed the functional significance of Lysine residues of putative sumoylation sites and the conserved ATP-binding motif of Smc5 by mutational analysis. In conclusion, our study highlights an important role for the SUMO ligase activity of Nse2 in maintaining genomic stability and suggests that sumoylation of Smc5 may be important for resisting MMS-induced genotoxic stress.

Structural Maintenance of Chromosomes (SMC) proteins are a highly conserved class of proteins required for the maintenance of genome stability and regulate nearly all aspects of chromosome biology. Eukaryotes, such as the budding yeast Saccharomyces cerevisiae, have six Smc proteins that form three SMC complexes in association with non-SMC proteins, i.e., the cohesin complex, the condensin complex and the Smc5/6 complex. The yeast Smc5/6 complex consists of Smc5, Smc6 and six non-Smc elements (Nse1-6) that are all essential for the survival of cells. Nse1 is the first non-smcelement that was identified associated with the Smc5/6 complex. Nse1 has a C-terminal RING-domain, which is a characteristic feature of some E3 ubiquitin ligases. A RING domain consists of eight conserved Zn-coordinating residues arranged in a cross-brace conformation. To understand the importance of this domain, we created site directed mutations in conserved residues identified by sequence alignment of the budding yeast Nse1 RING domain with that of other species. We found a new RING domain mutant nse1-103that was temperature sensitive at 37°C and showed an increased sensitivity towards genotoxic agents such as hydroxyurea (HU), methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. Thense1-103 mutant cells are slow growing and show delayed chromosomal replication at the restrictive temperature. Genetic interactions with replication factors such as RRM3, TOF1 etc. revealed thatnse1-103shows a synthetic sick growth defect in combination with rrm3∆ that is partially suppressed by deletion of TOF1. We found an enhancement in chromosome loss in nse1-103 compared to wild type cells. This was accompanied by a slight reduction in cohesion between the sister chromatids in nse1-103,suggesting a plausible mechanism for the chromosome destabilization observed in the mutant. Since Nse1 forms part of a trimeric sub-complex with Nse3 and Nse4 in the Smc5/6 complex, we performed a yeast two hybrid assay to test the interaction of nse1-103 with Nse3 or Nse4, and found a defect in interaction of nse1-103 with Nse3 and Nse4. In addition, a defect in association of nse1-103 with Smc5 or Smc6 could be observed by performing co-immunoprecipitation from yeast cell lysates, suggesting that the integrity of the RING-domain is critical for the interaction of Nse1 with other subunits of the Smc5/6 complex. However, there was no defect in the interaction between Nse3 and Smc5 in nse1-103, indicating that the interaction of these components within the complex isindependent of Nse1. We also identified a novel sequence motif near the RING domain of Nse1, deletion of which leads to an increased sensitivity towards genotoxic stressors and higher temperature. Biochemical characterization of this mutant also suggests a defect ininteraction with Nse3 or Nse4, and also with Smc5. The nse1 mutants also showed defects in post translational modification of Smc5 and other proteins. Since the Smc5/6 complex also has a SUMO E3 ligase, Mms21/Nse2, we also investigated genetic interactions between the RING domain mutant,nse1-103 and the SUMO ligase RING domain defective mutant,mms21∆sl, and found an exacerbation of the drug sensitive phenotypes in thense1-103 mms21∆sl double mutant relative to either of the single mutants nse1-103 or mms21∆sl, indicating that the two proteins contribute independently to the function of Smc5/6 complex in resisting genotoxic stress. In conclusion, the present study emphasizes the role of the RING domain of budding yeast Nse1 in resisting genotoxic stress and maintaining chromosome stability and reveals that the integrity of the RING-domain is critical for interactions of Nse1 with Nse3 and other Smc5/6 complex components. In addition, we report identification of another novel sequence motif in Nse1 that is also crucial for its interaction with other subunits of the Smc5/6 complex and for maintenance of post-translational modifications of some cellular proteins.

Les membres de la famille SMC (Structural Maintenance of Chromosomes), présents dans tous les domaines de la vie, sont impliqués dans des processus allant de la cohésion des chromatides-sœurs jusqu’à la réparation de l’ADN. Chacun des membres de cette famille, composée de 6 membres (Smc1 à Smc6), s’associe avec un autre membre ainsi qu’à des sous-unités non-SMC pour former 3 complexes : cohésine, condensine et Smc5-6. L’implication du complexe Smc5-6 dans plusieurs aspects du maintien de l’intégrité génomique est bien démontrée. Néanmoins, une question fondamentale concernant ce complexe demeure encore sans réponse: comment peut-il être impliqué dans autant d’aspects de la vie d’une cellule? Encore à ce jour, il est difficile de répondre à cette question en raison du manque d’information disponible au sujet des activités biochimiques de ce complexe. C’est pourquoi l’objectif de ce travail consiste en la caractérisation biochimique du complexe Smc5-6. La biochimie de cohésine et condensine suggère diverses possibilités en ce qui a trait aux activités biochimiques du complexe Smc5-6. La première étape de mon projet fut donc d’élaborer une procédure pour la purification de Smc5 et Smc6 après surexpression en levure. Après plusieurs expériences, il apparut clair que les deux protéines possèdent une activité de liaison à l’ADN simple brin (ADNsb) ainsi qu’à l’ADN double brins (ADNdb) et que, même si les protéines peuvent se lier aux deux types d’ADN, elles possèdent une plus grande affinité pour l’ADNsb. De plus, ces expériences permirent de démontrer que l’interaction entre Smc5 ou Smc6 et l’ADNsb est très stable, alors que l’interaction avec l’ADNdb ne l’est pas. Suite à l’obtention de ces résultats, la seconde étape fut la détermination de la ou des partie(s) de Smc5 et Smc6 permettant la liaison à l’ADN. Pour répondre à cette question, une dissection moléculaire fut réalisée, suivi d’une caractérisation des différents domaines constituants Smc5 et Smc6. De cette façon, il fut possible de démontrer qu’il existe deux sites de liaison à l’ADN sur Smc5 et Smc6 ; le premier site se trouvant dans le domaine «hinge» ainsi que dans la région adjacente du domaine «coiled-coil» et le second au niveau de la tête ATPase des deux protéines. Bien que les deux domaines puissent lier l’ADNsb, il fut démontré qu’une différence majeure existe au niveau de leur affinité pour ce type d’ADN. En effet, le domaine «hinge» possède une affinité plus forte pour l’ADNsb que la tête ATPase. De plus, cette dernière est incapable de lier l’ADNdb alors que le domaine «hinge» le peut. L’identification des sites de liaison à l’ADN sur Smc5 et Smc6 permettra de créer de nouveaux mutants possédant un défaut dans la liaison à l’ADN. Ainsi, l’étude du complexe Smc5-6 durant la réparation de l’ADN in vivo sera facilité. / The Smc5-6 complex is part of the SMC (Structural Maintenance of Chromosomes) family and is involved in the maintenance of genome integrity. This complex is required for the replication and repair of DNA. Unfortunately, the DNA substrates recognized by the Smc5-6 complex are still unknown. To address this gap, I used a biochemical approach to purify and functionally characterize the core of the Smc5-6 complex represented by the two SMC proteins. Subsequently, I wanted to understand which part(s) of Smc5 or Smc6 mediate their binding to DNA. I show here that Smc5 and Smc6 bind to all types of DNA tested. Despite this ability to associate with several types of nucleic acids, they have a clear preference for single-stranded DNA (ssDNA). The ability of Smc5 and Smc6 to link DNA independently of each other suggests that both SMC proteins have the potential to target the Smc5-6 complex to its DNA substrates in vivo. Furthermore, the minimal length of ssDNA required for the binding of Smc5 or Smc6 is between 45 to 75 nucleotides. This length of ssDNA is shorter than the size of ssDNA intermediates created during DNA repair or replication reactions. In addition to having a preference for ssDNA, the binding of both SMC proteins to this type of DNA is stronger than their binding to double-stranded DNA (dsDNA). Finally, the molecular dissection of SMC proteins into functional domains revealed that there are two independent DNA-binding sites on each molecule of Smc5 or Smc6. The first region is located in the hinge domain, while the second region is located in the ATPase head of the protein. The affinity and selectivity of independent domains towards DNA substrates suggest a functional differentiation between the two DNA-binding sites of SMC molecules. Indeed, the hinge domain has a greater affinity for ssDNA than the ATPase head. In terms of selectivity, the hinge domain is capable of binding to dsDNA whereas the ATPase head cannot. Taken together, our identification of the DNA-binding domains on Smc5 and Smc6 will enable the creation of new mutants with a defect in their DNA-binding activity. Thus, the study of the Smc5-6 complex during DNA repair, in vivo, will be facilitated.