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.

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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

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.