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Restriction endonucleases are a group of enzymes that cleave DNA at or around specific sequences, which are typically palindromic. A fosmid library was constructed from a metagenome isolated from soil from the Kogelberg Nature Reserve, Western Cape and was functionally screened for restriction endonucleases. Next-generation (NGS) Illumina sequencing technology was used to identify putative endonucleases. The sequence data generated was assembled and analysed using CLC Bio Genomics Workbench and bioinformatics tools (NCBI BLAST, REBASE and MG-RAST). Using these tools, genes encoding restriction-modification systems and endonuclease homologues were discovered. Three genes were identified and were recombinantly produced in Rosetta™ (DE3) pLysS and purified with IMAC using Ni-TED resin and subsequently characterised. These three genes were selected based on the identity percentage when compared to sequences on the NCBI database. Production of Endo8 was scaled up using 2 l fermenter and the purification done using ÄKTA Avant 150 FPLC using a HiScale 50 column packed with Ni-TED resin and the total amount of protein achieved was 58.82 mg.g-1. The productivity achieved at 17 hours (8 h harvest) was 2-fold greater than at 12 hours. Endonuclease activity of endo8 and endo52 was tested, both exhibited strong non-specific activity at 37 °C with an incubation period of 30 min. This work demonstrates that environmental soil samples are a valuable source for discovery of novel enzymes and also the utility of functional metagenomics to discover and purify these enzymes. These endonucleases may contribute to the next generation of reagent enzymes for molecular biology research. / Chemistry / M. Sc. (Life Sciences)

EcoR124I is a Type I restrictionmodification (RM) enzyme and as such forms multifunctional pentameric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on the motor subunit HsdR. When non-methylated invading DNA is recognized by the complex, two HsdR endonuclease/motor subunits start to translocate dsDNA without strand separation activity up to thousands base pairs towards the stationary enzyme while consuming ~1 molecule of ATP per base pair advanced. Whenever translocation is stalled the HsdR subunits cleave the dsDNA nonspecifically far from recognition site. The X-ray crystal structure of HsdR of EcoR124I bound to ATP gave a first insight of structural/functional correlation in the HsdR subunit. The four domains within the subunit were found to be in a square planer arrangement. Computational modeling including molecular dynamics in combination with crystallography, point mutations, in vivo and in vitro assays reveals how interactions between these four domains contribute to ATP-dependent DNA translocation, DNA cleavage or inter-domain communication between the translocase and endonuclease activities.

Mon projet a porté sur la caractérisation du mécanisme moléculaire des enzymes d'arenavirus (une 3'-5' exoribonucléase et une endonuclease) impliquées dans l'inhibition de la réponse innée IFN de type I et dans le vole de coiffe respectivement, et le développement d'une stratégie thérapeutique basée sur leur structures. Premièrement, j'ai résolu deux structures cristallographiques à haute résolution du domaine exoribonucléases du virus Mopeia (NP-exo MOPV) -un homologue du virus Lassa pathogène- en complexe avec deux ions différents. Ensuite, j'ai effectué une caractérisation fonctionnelle de l’activité exoribonucléase 3'-5' codée par ce domaine. Une corrélation entre la structure et la fonction de NP-exo MOPV démontre que; L’activité exoribonucléase 3'-5' est conservée chez les arenavirus pathogènes ainsi que chez les non-pathogènes. J'ai démontré pour la première fois que l'exoribonucléase est capable d'exciser un ARN misapparié, suggérant ainsi une potentielle activité de correction d'erreur par cette enzyme. Avec la structure de NP-exo MOPV, j'ai développé une stratégie in silico pour identifier des inhibiteurs potentiels spécifiques contre son activité et un inhibiteur a était identifié.En parallèle, nous avons résolu deux structures cristallographiques du domaine de l'endonuclease du virus de la LCMV en complexe avec deux ions catalytiques et deux composés appartenant a la famille des diketo. En résumé, ce travail éclaircit le rôle des exoribonucléases de la famille d'Arenaviridae allant de l’évasion de l'immunité innée à son implication directe dans la réplication. Il ouvre également la voie au développement des inhibiteurs contre ces nucléases. / My PhD work focused on the characterization of the molecular mechanism of two arenavirus enzymes - a 3'-5' exoribonuclease and an endonuclease - implicated in type I IFN suppression and mRNA cap-snatching respectively and the design of a structure based-drug strategy against them. First I solved two high resolution crystal structures of the exoribonuclease domain of Mopeia virus (NP-exo MOPV) -a non pathogenic homologue of the highly pathogenic Lassa virus- in complex with different metal ions. Next I performed an in depth functional characterization of the 3'-5' exoribonuclease activity encoded by this domain. By correlating the structure and function of NP-exo MOPV, I showed that; the 3'-5' exoribonuclease activity is conserved in pathogenic as well as in non-pathogenic arenaviruses. Also, I showed for the first time that this enzyme is able to excise a mismatched RNA suggesting that, arenaviruses might posses a mechanism to limit error incorporation by the RdR polymerase during replication. Using the crystal structure of NP-exo MOPV I designed a structure-based strategy to identify potential inhibitors specific for the 3'-5' exoribonuclease activity and have identified a potential inhibitor.Alongside, we solved two crystal structures of the endonuclease domain of LCMV in complex with two catalytic ions and two compounds belonging to the diketo family.In conclusion, this work has a deep implication extending the role of the Arenaviridae exoribonuclease from innate immunity evasion to direct implication in replication. It also paves the way for the development of inhibitors against these arenavirus nucleases.

Human mitochondrial DNA (mtDNA) is located in discrete DNA-protein complexes, so called nucleoids. These structures can be easily visualized in living cells by utilizing the fluorescent stain PicoGreen®. In contrary, cells devoid of endogenous mitochondrial genomes (ρ0 cells) display no mitochondrial staining in the cytoplasm. A modified restriction enzyme can be targeted to mitochondria to cleave the mtDNA molecules in more than two fragments, thereby activating endogenous nucleases. By applying this novel enzymatic approach to generate mtDNA-depleted cells the destruction of mitochondrial nucleoids in cultured cells could be detected in a time course. It is clear from these experiments that mtDNA-depleted cells can be seen as early as 48 h post-transfection using the depletion system. To prove that mtDNA is degraded during this process, mtDNA of transfected cells was quantified by real-time PCR. A significant decline could be observed 24 h post-transfection. Combination of both results showed that mtDNA of transfected cells is completely degraded and, therefore, ρ0 cells were generated within 48 h. Thus, the application of a mitochondrially-targeted restriction endonuclease proves to be a first and fast, but essential step towards a therapy for mtDNA disorders.

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The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

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