Abstract The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell. The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity. The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication. Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.

No description available.

Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).

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No description available.

Covalent modifications of histones play a critical role in many important biological processes such as transcription, DNA repair and recombination. Among the major modifications known so far, histone H3 acetylation at lysines 9 and 14 (H3K9/14), monoubiquitination of histone H2B at lysine123 (H2BK123) and H3 lysine 4 methylation (H3K4) are among the more studied ones. The importances of these modifications have been further stressed by its connection to various human diseases including cancers. Previous biochemical studies have shown that H2BK123 ubiquitination is mandatory for methylation at histone H3K4. However, little is known about the regulatory mechanisms of H3K4 methylation by H2B ubiquitination in vivo. Thus, the prime focus of this study is to understand the factors involved in the regulation of H2B ubiquitination, the regulatory mechanisms of the cross-talk between H2BK123 ubiquitination and H3K4 methylation and the role of these covalent modifications in transcriptional regulation under physiological conditions. Here in this study, I have shown that Ubp8p, a histone deubiquitinase, is a bona fide subunit of SAGA (Spt3-Ada-Gcn5 acetyltransferase) co-activator complex and selectively regulates both di and trimethylation of histone H3K4 at the core promoter of a SAGA-dependent gene in vivo. However, over the open reading frames for a subset of constitutive genes H2B ubiquitination selectively upregulates only H3K4 trimethylation but not dimethylation. Moreover, such an upregulation of H3K4 trimethylation has no impact on the RNA Polymerase II (RNAPII) recruitment and hence transcription of the respective genes. Interestingly, at an inducible gene, histone H2B ubiquitination promotes transcription elongation independently of H3K4 methylation. Furthermore, this study also demonstrates for the first time, the molecular mechanism for the cross-talk between H2B ubiquitination and H3K4 methylation in vivo. Evidently a COMPASS subunit, Cps35p, is necessary for the trans-tail cross talk between histones H2B and H3. Finally, this study also shows that Sgf73p, a SAGA subunit, is required for SAGA recruitment at the promoters of several SAGA dependent genes and facilitates transcription in both HAT-dependent and HAT-independent manner. Collectively, the results from this study not only provide deep insights into the regulatory mechanisms of H2B ubiquitination and H3K4 methylation (and their role in transcription) but also give a new functional dimension to SAGA subunit, Sgf73p, under physiological conditions. Given the role of histone acetylation, ubiquitination and methylation in many human diseases, the results from this study is of tremendous clinical value unveiling new therapeutical targets.

mRNA molecules form an intermediate in the transfer of sequences from DNA to ribosomes in order to guide protein production. Errors can be introduced into mRNA, producing aberrant proteins which place a strain on cellular regulatory machinery, causing increased risks of apoptosis, cancer, and decreased fitness. These errors may be introduced due to decreased transcriptional proofreading capabilities, exposure to chemicals, or mistakes in RNA editing machinery. It is important to investigate these causes of transcription errors to better understand the long-neglected area of mRNA fidelity which has such significant impacts on our cellular functions. In this paper, it was determined that addition of adenine opposite from abasic sites, not genomic uracil pairing with adenine, are a probable cause of G-to-A transcription errors. That exposure to Roundup causes increased levels of transcription errors, potentially due to oxidative stress. And finally, that off-target ADAR gene editing of transcripts occurs at high levels.

No description available.

Chez Saccharomyces cerevisiae, les souches mutantes pour Rrd1, une protéine qui possède une activité de peptidyl prolyl cis/trans isomérase, montrent une résistance marquée à la rapamycine et sont sensibles au 4-nitroquinoline 1-oxide, un agent causant des dommages à l’ADN. PTPA, l’homologue de Rrd1 chez les mammifères, est reconnu en tant qu’activateur de protéine phosphatase 2A. Notre laboratoire a précédemment démontré que la surexpression de PTPA mène à l’apoptose de façon indépendante des protéines phosphatase 2A. La fonction moléculaire de Rrd1/PTPA était encore largement inconnue au départ de mon projet de doctorat. Mes recherches ont d’abord montré que Rrd1 est associé à la chromatine ainsi qu’à l’ARN polymérase II. L’analyse in vitro et in vivo par dichroïsme circulaire a révélé que Rrd1 est responsable de changements au niveau de la structure du domaine C-terminal de la grande sous-unité de l’ARN polymérase II, Rpb1, en réponse à la rapamycine et au 4-nitroquinoline 1-oxide. Nous avons également démontré que Rrd1 est requis pour modifier l’occupation de l’ARN polymérase II sur des gènes répondant à un traitement à la rapamycine. Finalement, nous avons montré que suite à un traitement avec la rapamycine, Rrd1 médie la dégradation de l’ARN polymérase II et que ce mécanisme est indépendant de l’ubiquitine. La dernière partie de mon projet était d’acquérir une meilleure connaissance de la fonction de PTPA, l’homologue de Rrd1 chez les mammifères. Nos résultats montrent que le «knockdown» de PTPA n’affecte pas la sensibilité des cellules à différentes drogues telles que la rapamycine, le 4-nitroquinoline 1-oxide ou le peroxyde d’hydrogène (H2O2). Nous avons également tenté d’identifier des partenaires protéiques pour PTPA grâce à la méthode TAP, mais nous ne sommes pas parvenus à identifier de partenaires stables. Nous avons démontré que la surexpression de la protéine PTPA catalytiquement inactive n’induisait pas l’apoptose indiquant que l’activité de PTPA est requise pour produire cet effet. Finalement, nous avons tenté d’étudier PTPA dans un modèle de souris. Dans un premier lieu, nous avons déterminé que PTPA était exprimé surtout au niveau des tissus suivants : la moelle osseuse, le thymus et le cerveau. Nous avons également généré avec succès plusieurs souris chimères dans le but de créer une souris «knockout» pour PTPA, mais l’allèle mutante ne s’est pas transférée au niveau des cellules germinales. Mes résultats ainsi que ceux obtenus par mon laboratoire sur la levure suggèrent un rôle général pour Rrd1 au niveau de la régulation des gènes. La question demeure toujours toutefois à savoir si PTPA peut effectuer un rôle similaire chez les mammifères et une vision différente pour déterminer la fonction de cette protéine sera requise pour adresser adéquatement cette question dans le futur. / In Saccharomyces cerevisiae, mutants devoid of Rrd1, a protein possessing in vitro peptidyl prolyl cis/trans isomerase activity, display striking resistance to rapamycin and show sensitivity to the DNA damaging agent 4-nitroquinoline 1-oxide. PTPA, the mammalian homolog of Rrd1, has been shown to activate protein phosphatase 2A. Our laboratory previously found that overexpression of PTPA leads to apoptosis independently of PP2A. At the outset of my thesis work, the molecular function of Rrd1/PTPA was largely unknown. My work has shown that Rrd1 is associated with the chromatin and interacts with RNA polymerase II. In vitro and in vivo analysis with circular dichroism revealed that Rrd1 mediates structural changes of the C-terminal domain of the large subunit of RNA pol II, Rpb1, in response to rapamycin and 4-nitroquinoline 1-oxide. Consistent with this, we demonstrated that Rrd1 is required to alter RNA pol II occupancy on rapamycin responsive genes. We also showed that upon rapamycin exposure Rrd1 mediates the degradation of RNA polymerase II and that this mechanism is ubiquitin-independent. Another part of my work was to gain insight into the function of PTPA, the mammalian counterpart of Rrd1. PTPA knockdown did not affect sensitivity to rapamycin, 4-nitroquinoline 1-oxide or H2O2. We also attempted to find protein interaction partners for PTPA using tandem affinity purification, but no stable partners for PTPA were found. We also demonstrated that overexpression of a catalytically inactive PTPA mutant did not induce apoptosis, indicating that PTPA activity is required to produce this effect. Finally, we attempted to study PTPA in a mouse model. We first determined that PTPA was expressed in a tissue-specific manner and was most abundant in bone marrow, thymus and brain. We pursued creation of a knockout mouse and successfully generated chimeras, but the mutated allele was not transmitted to the germline. My data and other data from our laboratory regarding the yeast work suggest a general role for Rrd1 in regulation of gene transcription. Whether PTPA has a similar function in mammalian cells remains unknown, and a different vision of what the protein does in mammalian cells will be required to adequately address this question in the future.

Le virus T lymphotropique humain HTLV-1 est l’agent étiologique de la leucémie-lymphome T de l’adulte (ATLL) et de nombreuses maladies inflammatoires. HTLV-1 est associée à de nombreuses modifications quantitatives de l’expression des gènes cellulaires. À ce jour, ces modifications ont été décrites essentiellement à l’échelle transcriptionnelle à travers notamment les effets de l’oncoprotéine virale Tax, et plus récemment HBZ. Outre leurs impacts sur les niveaux d’activité des promoteurs, certains facteurs apparaissent jouer également un rôle dans la régulation de l’épissage alternatif. Ce mécanisme essentiel à la diversité du transcriptome et du protéome cellulaire, apparait étroitement couplé à la transcription et ses dérégulations sont de plus en plus décrites dans les phénomènes cytotoxiques et pathogènes tels que les infections et les cancers. Dans ce contexte, mon travail s’est intéressé à caractériser les profils d’expression des exons des cellules T CD4+ infectées ou non, et transformée ou non par HTLV-1 in vivo. Dans une seconde étude, j’ai abordé les aspects mécanistiques des modifications d’épissage alternatif par HTLV-1. Mes données montrent que, outre ses effets sur la régulation quantitative de l’expression des gènes cellulaires, l’activation de la voie NF-kB par l’oncogène Tax est impliquée dans la reprogrammation de l’épissage alternatif de nombreux gènes. Ces données révèlent un nouveau degré de complexité dans les mécanismes de dérégulation de l’expression des gènes cellulaires par HTLV-1 et ouvre de nouvelles perspectives d’investigations dans la compréhension des processus leucémogènes associés à l’infection par le virus HTLV-1 / Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects. HTLV-1 quantitatively modifies the pattern of cellular gene expression. Exon expression analysis shows that patients’ untransformed and malignant HTLV-1+ CD4+ T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. A total of 486 exon modifications occurred in untransformed infected CD4+ cells were detected in ATLL arguing for a role of AEUs in HTLV-1 leukemogenesis. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patters of 3977 exons that discriminate uninfected, infected, and transformed CD4+ T-cells. Exposing cells to splicing inhibitors revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provides news targets for treating ATLL. Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets