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DNA damage occurs to all living things; its subsequent repair is a crucial component of life. The most dangerous, and potentially most useful form of DNA damage is the double strand break (DSB). A DSB is defined by breaks occurring to both sugar phosphate backbones in close enough proximity that they lead to the separation of the two pieces of the DNA. This type of damage will kill the cell if left unrepaired. It is the most lethal type of DNA damage. Most living organisms have also developed ways to take advantage of DSBs through their repair systems, primarily as a means of introducing genetic variation. There are two primary DSB repair pathways across life: homologous recombination (HR) and non-homologous end-joining (NHEJ). The focus of this work is NHEJ. NHEJ is known as “error-prone” because it does not use a homologous template and can introduce small addition or deletion mutations during the repair process. This pathway has been extensively studied in eukaryotes and is known as the primary form of DSB repair in mammalian cells, however the prokaryotic NHEJ system was more recently identified and as a result, a void of information surrounds it. NHEJ is comprised of 3 core steps: DSB recognition and binding, DNA end processing, and ligation. In the eukaryotic version of NHEJ these 3 steps involve a plethora of factors; conversely, in the prokaryotic version, the same functionality is accomplished by just 2 proteins, bacterial Ku and LigD. The focus of this research is Ku: the DNA end-binding protein responsible for identifying the DSB, binding and protecting the DNA end, as well as recruiting LigD to the break. Ku is composed of 2 domains, the first of which is predicted to be highly homologous to eukaryotic Ku’s equivalent domain; this is the core domain which forms a ring-like structure that DNA threads through. The second is completely unique to bacterial Ku, it is the C-terminal domain, which can further be split into 2 sub-domains, the minimal C-terminus, and the extended C-terminus. The sub-domains are defined by their level of conservation across bacterial species, with the minimal C-terminus being highly conserved, while the extended C-terminus is highly variable. Using DNA-binding assays and several mutant constructs which affect the C-terminal domain, I show that this C-terminus is unexpectedly responsible for destabilizing the Ku-DNA interaction. This observation leads me to hypothesize that maintaining a weak interaction with DNA is important for Ku because of the other proteins which need access to the DNA (e.g. replicative helicase). While Ku is bound, it could be capable of blocking regions of DNA, in turn blocking other vital cellular processes like replication. Ku maintaining a lower affinity for DNA should facilitate Ku displacement by other proteins. A tighter binding would restrict Ku’s freedom to move on DNA making it more likely to inhibit other critical pathways. To better understand Ku, I attempted to solve the Ku structure using X-ray crystallography, and was able to achieve crystals of Ku, however diffraction was too limited for a structure. Another way to investigate the validity of my proposed model is to use a biophysical approach with atomic force microscopy (AFM) to visualize protein-DNA complexes. The initial work has established key controls for future Ku-DNA AFM work by imaging and analyzing Ku on its own. Interest in bacterial NHEJ is two-fold from the antimicrobial perspective: NHEJ is a highly mutagenic pathway, so it serves as a proverbial well for differentiation and thus the development of antimicrobial resistance (AMR); NHEJ is very important in bacteria that enter a stationary phase due to their lack of a homologous piece of DNA for HR. Thus, NHEJ inhibition could be useful for slowing bacterial evolution and potentially as a treatment for infections such a Mycobacterium tuberculosis, which is known to lie dormant in host macrophages for long periods of time. To investigate the viability of NHEJ inhibition, I had begun the process of creating ∆ku strains of Pseudomonas aeruginosa to simulate Ku inhibition under various conditions. This Ku project is the focus of the first two chapters, however, during my Master’s degree I participated in 2 other major projects. The third chapter details a bacterial DNA damage tolerance pathway, which similarly is highly mutagenic and poorly characterized: the ImuABC translesion synthesis polymerase complex. The fourth and final chapter details the work for a Journal of Visualized Experiments article meant to highlight the benefits of AFM as a means of studying protein-DNA interactions. / Thesis / Master of Science (MSc)

DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations. / Ph. D.

The teratogenic effects of valproic acid (VPA) are well known, though its teratogenic mechanism remains unknown. VPA induces oxidative stress, which may lead to double strand breaks (DSBs) in DNA. Though the cell may repair this damage via homologous recombination (HR) and non-homologous end joining (NHEJ), repair is not always error-free; genomic instability may arise from gene deletions, amplifications, rearrangements, and loss of heterozygosity. Such alterations may underpin VPAʼs teratogenicity. The present study evaluated VPAʼs ability to induce NHEJ and HR and characterized the changes in expression of two proteins key to HR (RAD51) and NHEJ (XRCC4). Using pKZ1 transgenic mice (C57BL/6 genetic background), we sought to measure NHEJ events via X-gal staining. Although consistent staining was observed in adult male brain (positive control), no staining was observed in embryos 12 or 24 hours after in utero exposure to a teratogenic dose of VPA (500 mg/kg, maternal subcutaneous dose) on gestational day 9 (GD9). To determine whether the lack of staining observed in embryos was due to low/absent expression of key DSB-repair proteins, we measured mRNA/protein expression of RAD51 and XRCC4 in C57BL/6, GD9-exposed embryos and maternal brain. One hour after treatment, XRCC4 was increased at the protein level in brain and embryo. RAD51 was not increased in embryos and not detected in adult brain. These data suggest that embryos do possess the protein mediators of NHEJ and HR and that VPA-induced changes in expression of XRCC4 may influence the type of repair pursued, potentially affecting DSB repair fidelity (accuracy). Determination of fidelity of VPA-induced HR was attempted with the Chinese hamster ovary cell line (CHO33) using DNA sequencing; low template concentration and purity precluded successful sequencing of DNA from recombinant colonies and the assessment of fidelity. Overall, these data demonstrate that the lack of X-gal staining observed in pKZ1 embryos is not due to an underexpression of at least one key protein in the NHEJ pathway. Furthermore, a VPA-induced change in the the type of repair pathway pursued by the embryo may have teratological implications. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-10-15 11:06:30.613

De nombreuses études montrent un rôle important de l'architecture du noyau sur la stabilité du génome. Les lamines sont les constituants majeurs de l’enveloppe nucléaire et sont impliquées dans de nombreux processus, notamment, la régulation génique, la réplication et le maintien de la structure du noyau. Il en existe 2 types, les lamines A/C et les lamines B. Certaines mutations des lamines A/C sont à l’origine de syndromes progéroïdes, classés jusqu’à présents en deux catégories : ceux associés à une dérégulation des lamines (laminopathies) et ceux associés à un défaut de réparation de l’ADN, dont l’Ataxie Télangiectasie (A-T). Il est proposé que le vieillissement prématuré observé dans les laminopathies est dû à un défaut de réparation de l’ADN, qui serait alors la voie commune d’induction de sénescence des syndromes progéroïdes. Ceci est appuyé par le fait que de plus en plus de données associent les mutations des lamines A/C à des défauts de réparation de l’ADN. La mise en évidence, par notre laboratoire d’une accumulation de lamine B1 dans A-T et dans deux autres syndromes progéroïdes, pose la question de l’impact de la surexpression de la lamine B1 sur la réparation de l’ADN, d’autant plus que de plus en plus de données associent une augmentation de la lamine B1 à de nombreux cancers, bien que le mécanisme moléculaire ne soit pas connu. Au cours de ma thèse, j’ai donc pu montrer, notamment à l’aide de substrats intra-Chromosomiques, qu’une surexpression de lamine B1 entraînait un défaut de réparation des cassures double-Brin par NHEJ associé à un défaut de recrutement de 53BP1 à la cassure. La mise en évidence d’une interaction entre 53BP1 et la lamine B1, rompue après dommages permet de suggérer un nouveau rôle de la lamine B1 comme réservoir de 53BP1, régulant son recrutement aux cassures. De plus, d’autres résultats suggèrent que la lamine B1 agirait également au niveau de la signalisation du dommage en altérant l’activation d’ATM par un mécanisme qu’il reste à caractériser. L’ensemble de ces résultats montrent un nouveau rôle très important de la lamine B1 dans la signalisation des dommages et la régulation du recrutement des protéines de réparation, ouvrant la voie à une meilleure compréhension de l’implication de la lamine B1 dans la sénescence et le cancer. / Many studies show an important role of nuclear shape on genome stability. Lamins are the major components of the nuclear envelope and are implicated in numerous processes like gene regulation, DNA replication and the maintenance of nuclear structure. There are 2 types of lamins : lamin A/C and lamin B. Some mutations of lamin A/C cause progeroid syndromes, which are classified untill now in two categories : those due to lamins deregulation and those due to DNA repair defects, including Ataxia Telangiectasia (A-T). Accelerated aging observed in laminopathies is proposed to be due to a DNA repair defect, which would be the common pathway leading to senescence in progeroid syndromes. This is supported by many data linking lamin A mutations to DNA repair defects. Our laboratory reported that lamin B1 accumulates in A-T and Fanconi and another study showed also an accumulation in Werner syndrome, which is another progeroïd syndrome. This discovery raises a question about the impact of lamin B1 overexpression on DNA repair, especially as more and more data show an increase of lamin B1 in several cancers, although the molecular mechanism is still unclear. During my thesis, I showed, in particular with intrachromosomal substrates, that lamin B1 overexpression leads to an NHEJ double-Strand break (DSB) repair defect associated with a defect of 53BP1 recruitment to the break. The discovery of an interaction between 53BP1 and lamin B1, which is broken after damage, suggests a new role of lamin B1 as a « reservoir » of 53BP1, regulating its recruitment to the break. In addition, we obtained results suggesting that lamin B1 could also act in the DSB signalisation pathway by affecting ATM activation through a mechanism that we still have to characterize.All together, these datas show a new important role of lamin B1 in DSB signalisation and in the regulation of the recruitment of repair proteins, paving the way to a better understanding of the implication of lamin B1 in senescence and cancer.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19047号 / 農博第2125号 / 新制||農||1032 / 31998 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当

L'hétérochromatine, de nature compacte et répétitive, limite l’accès à l'ADN et fait de la réparation des DSBs un processus difficile que les cellules doivent surmonter afin de maintenir leur intégrité génomique. Pour y étudier la réparation des DSBs, nous avons conçu un système CRISPR / Cas9 dans lequel les DSB peuvent être efficacement et spécifiquement induites dans l'hétérochromatine de fibroblastes de souris NIH3T3. En développant un système CRISPR / Cas9 hautement spécifique et robuste pour cibler l'hétérochromatine péricentrique, nous avons montré que les DSB en G1 sont positionnellement stables et réparés par NHEJ. En S / G2, ils se déplacent vers la périphérie de ce domaine pour être réparés par HR. Ce processus de relocalisation dépend de la résection et de l'exclusion de RAD51 du domaine central de l'hétérochromatine. Si ces cassures ne se relocalisent pas, elles sont réparées dans le cœur du domaine de l'hétérochromatine par NHEJ ou SSA. D'autre part, les DSBs dans l'hétérochromatine centromérique activent NHEJ et HR tout au long du cycle cellulaire. Nos résultats révèlent le choix de la voie de réparation différentielle entre l'hétérochromatine centromérique et péricentrique, ce qui régule également la position des DSBs. / Heterochromatin is the tightly packed form of repetitive DNA, essential for cell viability. Its highly compacted and repetitive nature renders DSB repair a challenging process that cells need to overcome in order to maintain their genome integrity. Developing a highly specific and robust CRISPR/Cas9 system to target pericentric heterochromatin, we showed that DSBs in G1 are positionally stable and repaired by NHEJ. In S/G2, they relocate to the periphery of this domain to be repaired by HR. This relocation process is dependent of resection and RAD51 exclusion from the core domain of heterochromatin. If these breaks fail to relocate, they are repaired within heterochromatin by NHEJ or SSA. On the other hand, DSBs in centromeric heterochromatin activate both NHEJ and HR throughout the cell cycle. Our results reveal the differential repair pathway choice between centromeric and pericentric heterochromatin that also regulates the DSB position.

L'hétérochromatine est une caractéristique conservée des chromosomes eucaryotes, avec des rôles centraux dans la régulation de l'expression des gènes et le maintien de la stabilité du génome. Comment la réparation de l'ADN est régulée par l'hétérochromatine reste mal compris. Chez Saccharomyces cerevisiae, le complexe SIR (Silent Information Regulator) assemble une fibre de chromatine compacte. La chromatine SIR limite la résection aux cassures double-brin (DSB) protégeant les extrémités chromosomiques endommagées contre la perte d'informations génétiques. Toutefois, lesquels des trois complexes de résection redondants, MRX-Sae2, Exo1 et Sgs1-Dna2 sont inhibés et par quel(s) mécanisme(s) reste à decouvrir. Nous montrons que Sir3, le facteur de fixation des histones de l’hétérochromatine de Saccharomyces cerevisiae, interagit physiquement avec Sae2 et inhibe toutes ses fonctions. Cette interaction limite notamment la résection médiée par Sae2, stabilise MRX à la DSB et augmente le Non-Homologous End Joining (NHEJ). De plus, la chromatine répressive SIR inhibe partiellement les deux voies de résection extensive médiées par Exo1 et Sgs1-Dna2 par des mécanismes distincts. L'inhibition par les SIR de la résection extensive et de Sae2 favorise la NHEJ et limite le Break-Induced Replication (BIR), prévenant ainsi de la perte d'hétérozygotie au niveau des subtélomères. / Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in regulation of gene expression and maintenance of genome stability. How DNA repair occurs in heterochromatin remains poorly described. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) complex assembles a compact chromatin fibre. SIR-mediated repressive chromatin limits Double Strand Break (DSB) resection protecting damaged chromosome ends against the loss of genetic information. However, which of the three redundant resection complexes, MRX-Sae2, Exo1 and Sgs1-Dna2 are inhibited and by which mechanism remains to be deciphered. We show that Sir3, the histone-binding factor of yeast heterochromatin, physically interacts with Sae2-mediated resection and inhibits all its functions. Notably, this interaction limits Sae2-mediated resection, delays MRX removal from DSB ends and promotes Non-Homologous End Joining (NHEJ). In addition, SIR-mediated repressive chromatin partially inhibits the two long range resection pathways mediated by Exo1 and Sgs1-Dna2 by distinct mechanisms. Altogether SIR mediated inhibition of extensive resection and of Sae2 promotes NHEJ and limits Break-Induced Replication (BIR) preventing loss of heterozygosity at subtelomeres.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19047号 / 農博第2125号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4929(農学部図書室) / 31998 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 喜多 恵子, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19401号 / 医博第4052号 / 新制||医||1012(附属図書館) / 32426 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙田 穣, 教授 平岡 眞寛, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM