Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.

Ce travail a consisté en la synthèse d’outils chimiques et au développement de leurs applications biologiques. Dans un premier temps, des sondes pour l’étude de la Topoisomérase IIA humaine ont été synthétisées. Ces sondes ont alors été testées sur lysat cellulaire pour la capture des protéines présentant une affinité pour ces médicaments. Dans un second temps, un nouveau lien clivable en conditions non dénaturantes pour des applications en protéomique chimique a été developpé. Ainsi, après optimisation de la structure, il a été intégré au sein d’une sonde d’affinité pour évaluer sa capacité de capture et libération de la PARP 1. Enfin, la réaction de click entre un azoture et un cyclooctyne a été appliquée à l’élimination d’une drogue circulante dans le sang.Après l’étude cinétique de la réaction, l’activité biologique et la pharmacocinétique des différents composés ont été évaluées pour optimiser la réaction de click in vivo. / The main goal of this work was to synthesize chemical tools and to developp their biological applications. In the first part, probes for the study of Topoisomerase II via chemical proteomic were synthesized. They were then used for pulldown experiments on cell lysats. In a second part, a new cleavable linker in non denaturing conditions was developped for chemical proteomic applications. After optimisation of the structure, it was incorporated in an affinity probe and tested for the pulldown of PARP 1. Finally, a click chemistry reaction, the SPAAC, was used to provok the elimination of a circulating drug. After the study of the kinetic of the reaction, the biological activity and the pharmacokinetic of the different compounds were evaluated to optimise the click reaction in vivo.

La division cellulaire est un processus fondamental des êtres vivants. À chaque division cellulaire, le matériel génétique d'une cellule mère est dupliqué et ségrégé pour produire deux cellules filles identiques; un processus nommé la mitose. Tout d'abord, la cellule doit condenser le matériel génétique pour être en mesure de séparer mécaniquement et également le matériel génétique. Une erreur dans le niveau de compaction ou dans la dynamique de la mitose occasionne une transmission inégale du matériel génétique. Il est suggéré dans la littérature que ces phénomènes pourraient causé la transformation des cellules cancéreuses. Par contre, le mécanisme moléculaire générant la coordination des changements de haut niveau de la condensation des chromosomes est encore incompris. Dans les dernières décennies, plusieurs approches expérimentales ont identifié quelques protéines conservées dans ce processus. Pour déterminer le rôle de ces facteurs dans la compaction des chromosomes, j'ai effectué un criblage par ARNi couplé à de l'imagerie à haute-résolution en temps réel chez l'embryon de C. elegans. Grâce à cette technique, j'ai découvert sept nouvelles protéines requises pour l'assemblage des chromosomes mitotiques, incluant la Ribonucléotide réductase (RNR) et Topoisomérase II (topo-II). Dans cette thèse, je décrirai le rôle structural de topo-II dans l'assemblage des chromosomes mitotiques et ces mécanismes moléculaires. Lors de la condensation des chromosomes, topo-II agit indépendamment comme un facteur d'assemblage local menant par la suite à la formation d'un axe de condensation tout au long du chromosome. Cette localisation est à l'opposé de la position des autres facteurs connus qui sont impliqués dans la condensation des chromosomes. Ceci représente un nouveau mécanisme pour l'assemblage des chromosomes chez C. elegans. De plus, j'ai découvert un rôle non-enzymatique à la protéine RNR lors de l'assemblage des chromosomes. Lors de ce processus, RNR est impliqué dans la stabilité des nucléosomes et alors, permet la compaction de haut niveau de la chromatine. Dans cette thèse, je rapporte également des résultats préliminaires concernant d'autres nouveaux facteurs découverts lors du criblage ARNi. Le plus important est que mon analyse révèle que la déplétion des nouvelles protéines montre des phénotypes distincts, indiquant la fonction de celles-ci lors de l'assemblage des chromosomes. Somme toute, je conclus que les chromosomes en métaphase sont assemblés par trois protéines ayant des activités différentes d'échafaudage: topoisomérase II, les complexes condensines et les protéines centromériques. En conclusion, ces études prouvent le mécanisme moléculaire de certaines protéines qui contribuent à la formation des chromosomes mitotiques. / Cell division is a fundamental process that continuously happens in all living organisms. In each cell division, genetic material of the parent cell duplicates and segregates to produce genetically identical daughter cells in a process called mitosis. Cells need to condense their genetic material to be able to partition them equally. Any subtle defects, either timing or compaction level, could lead to the unequal inheritance of genetic material, a phenomenon that is believed to be the leading cause of cancerous transformation. However, the precise molecular mechanisms underlying the coordinated changes of higher-order chromosome structure are poorly understood. In the last two decades, various approaches have identified several conserved factors required for chromosome condensation. To define the roles of known and novel factors in this process, I performed an RNAi based screen using high-resolution live imaging of the C. elegans one-cell embryo. Importantly, using an in vivo approach, I discovered seven novel factors required for mitotic chromosome assembly, including Ribonulceotide reducatase (RNR) and DNA topoisomerase II (topo-II). In this thesis, I report a structural role for topo-II in mitotic chromosome assembly and underlying molecular mechanisms. During chromosome condensation process, topo-II acts independently as a local assembly factor leading to global chromosome axis formation, contradicting models that chromosomes organize around preassembled scaffolds, thus representing a novel pathway for chromosome assembly in C. elegans. Furthermore, I also discovered a non-enzymatic role of RNR in the mitotic chromosome assembly process. During this process, RNR is involved in nucleosome stability, and thereby, it allows higher-order chromatin assembly. In this thesis, I also report preliminary data for other novel factors that I discovered in the RNAi based screen for factors involved in chromosome condensation. Importantly, my analyses revealed that the depletion of several proteins results in distinct chromosome condensation phenotypes, indicating that they function in discrete events during mitotic chromosome assembly. In sum, I conclude that metaphase chromosomes are built by the distinct scaffolding activities of three proteins: DNA topoisomerase II, condensin complexes and centromere proteins. Taken together, these studies provide underlying molecular mechanisms contributing to the mitotic chromosome formation.

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