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L'ADN de chaque cellule est constamment soumis à des stress pouvant compromettre son intégrité. Les bris double-brins sont probablement les dommages les plus nocifs pour la cellule et peuvent être des sources de réarrangements chromosomiques majeurs et mener au cancer s’ils sont mal réparés. La recombinaison homologue et la jonction d’extrémités non-homologues (JENH) sont deux voies fondamentalement différentes utilisées pour réparer ce type de dommage. Or, les mécanismes régulant le choix entre ces deux voies pour la réparation des bris double-brins demeurent nébuleux. Le complexe Mre11-Rad50-Xrs2 (MRX) est le premier acteur à être recruté à ce type de bris où il contribue à la réparation par recombinaison homologue ou JENH. À l’intersection de ces deux voies, il est donc idéalement placé pour orienter le choix de réparation. Ce mémoire met en lumière deux systèmes distincts de phosphorylation du complexe MRX régulant spécifiquement le JENH. L’un dépend de la progression du cycle cellulaire et inhibe le JENH, tandis que l’autre requiert la présence de dommages à l’ADN et est nécessaire au JENH. Ensembles, nos résultats suggèrent que le complexe MRX intègre différents phospho-stimuli pour réguler le choix de la voie de réparation. / The genome of every cell is constantly subjected to stresses that could compromise its integrity. DNA double-strand breaks (DSB) are amongst the most damaging events for a cell and can lead to gross chromosomal rearrangements, cell death and cancer if improperly repaired. Homologous recombination and non-homologous end joining (NHEJ) are the main repair pathways responsible for the repair of DSBs. However, the mechanistic basis of both pathways is fundamentally different and the regulation of the choice between both for the repair of DSBs remains largely misunderstood. The Mre11-Rad50-Xrs2 (MRX) complex acts as a DSB first responder and contributes to repair by both homologous recombination and NHEJ. Being at the crossroads of both DSB repair pathways, the MRX complex is therefore in a convenient position to influence the repair choice. This thesis unravels two distinct phosphorylation systems modifying the MRX complex and specifically regulating repair by NHEJ. The first relies on cell cycle progression and inhibits NHEJ, while the second requires the presence of DNA damage and is necessary for efficient NHEJ. Together, our results suggest a model in which the MRX complex would act as an integrator of phospho-stimuli in order to regulate the DSB repair pathway choice.

L'ADN de chaque cellule est constamment soumis à des stress pouvant compromettre son intégrité. Les bris double-brins sont probablement les dommages les plus nocifs pour la cellule et peuvent être des sources de réarrangements chromosomiques majeurs et mener au cancer s’ils sont mal réparés. La recombinaison homologue et la jonction d’extrémités non-homologues (JENH) sont deux voies fondamentalement différentes utilisées pour réparer ce type de dommage. Or, les mécanismes régulant le choix entre ces deux voies pour la réparation des bris double-brins demeurent nébuleux. Le complexe Mre11-Rad50-Xrs2 (MRX) est le premier acteur à être recruté à ce type de bris où il contribue à la réparation par recombinaison homologue ou JENH. À l’intersection de ces deux voies, il est donc idéalement placé pour orienter le choix de réparation. Ce mémoire met en lumière deux systèmes distincts de phosphorylation du complexe MRX régulant spécifiquement le JENH. L’un dépend de la progression du cycle cellulaire et inhibe le JENH, tandis que l’autre requiert la présence de dommages à l’ADN et est nécessaire au JENH. Ensembles, nos résultats suggèrent que le complexe MRX intègre différents phospho-stimuli pour réguler le choix de la voie de réparation. / The genome of every cell is constantly subjected to stresses that could compromise its integrity. DNA double-strand breaks (DSB) are amongst the most damaging events for a cell and can lead to gross chromosomal rearrangements, cell death and cancer if improperly repaired. Homologous recombination and non-homologous end joining (NHEJ) are the main repair pathways responsible for the repair of DSBs. However, the mechanistic basis of both pathways is fundamentally different and the regulation of the choice between both for the repair of DSBs remains largely misunderstood. The Mre11-Rad50-Xrs2 (MRX) complex acts as a DSB first responder and contributes to repair by both homologous recombination and NHEJ. Being at the crossroads of both DSB repair pathways, the MRX complex is therefore in a convenient position to influence the repair choice. This thesis unravels two distinct phosphorylation systems modifying the MRX complex and specifically regulating repair by NHEJ. The first relies on cell cycle progression and inhibits NHEJ, while the second requires the presence of DNA damage and is necessary for efficient NHEJ. Together, our results suggest a model in which the MRX complex would act as an integrator of phospho-stimuli in order to regulate the DSB repair pathway choice.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.

Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques.  This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.

Indiana University-Purdue University Indianapolis (IUPUI) / In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.

Indiana University-Purdue University Indianapolis (IUPUI) / Translation Initiation in protein synthesis is a crucial step controlling gene expression that enhanced by eukaryotic translation initiation factors (eIFs). eIF3a, the largest subunit of eIF3 complexes, has been shown to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of proteins important for repair of double strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a up-regulation sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) activity by suppressing expression level of NHEJ repair proteins such as DNA-PKcs and vice versa. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies including ionizing radiation.