京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18879号 / 医博第3990号 / 新制||医||1008(附属図書館) / 31830 / 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 小松 賢志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Genome editing is an exciting field that allows for the precise modification of an organism's DNA. One of the most advanced tools in this area is CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), which creates a DSB (Double-strand break) at a specific location in the genome. This break can then be repaired by the cell using one of two pathways – NHEJ (nonhomologous end joining) or HDR (homology-directed repair) HDR leads to more precise repair and is used to create KI (Knock-In) modifications by introducing a homologous piece of DNA with the desired changes. However, HDR is a rare event that competes with the error prone NHEJ pathway, limiting its efficiency. HDR mainly occurs in the G2 and S phases of the cell cycle, making it a challenge to control and target. To improve KI efficiency, researchers have used strategies such as inhibiting NHEJ or activating HDR. This study focuses on identifying direct and indirect activators of HDR through a library assay screening. We established a robust method for screening compounds in HEK293 cells that relies on a plasmid-based delivery Cas9, gRNA (guide RNA), and synthetic ssDNA (single strand DNA). Out of 3,000 compounds screened, 1% showed a higher signal than the positive control, and approximately 10% presented a higher signal than untreated cells. The top 5 compounds were further validated in dose response. Our system opens new avenues for improving the efficiency of KI modifications.

No description available.

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)

L’élimination programmée d’ADN spécifique de la lignée germinale pour former un nouveau noyau somatique a été décrite chez les eucaryotes. Ces réarrangements sont initiés par l’introduction de cassures double brin (CDB) de l’ADN et la préservation de l’intégrité du génome requiert une réparation efficace. Chez Paramecium tetraurelia, le génome est largement réarrangé pendant le développement du nouveau noyau somatique, après l’introduction de milliers de cassures double brin programmées par la transposase domestiquée PiggyMac (Pgm)Ces réarrangements consistent en l’excision précise de dizaines de milliers de séquences uniques et non codantes (IES) qui interrompent 47% des gènes dans la lignée germinale ; et l’élimination hétérogène de séquences répétées qui mène à des délétions internes de taille variable ou à la fragmentation des chromosomes avec addition de télomères aux extrémités.L’implication de la voie du Non Homologous End Joining (NHEJ) dans l’excision précise des IES a été prouvée. Dans des cellules déplétées de Ligase IV ou XRCC4, les cassures aux bornes des IES sont introduites normalement mais il n’y a pas de jonctions d’excision formées et les extrémités cassées s’accumulent sans être dégradées. Mais la voie de réparation impliquée dans les réarrangements imprécis est encore inconnue. L’hypothèse d’une réparation par la voie NHEJ alternative (alt-NHEJ), indépendante de Ku et impliquant la résection des extrémités et l’utilisation de microhomologie, a été émise. C’est pourquoi pendant ma thèse je me suis intéressé à ma thèse au rôle des protéines Ku.Deux gènes KU70 et trois gènes KU80 ont été identifiés dans le génome de la paramécie. KU70a et KU80c sont spécifiquement induits pendant les réarrangements programmés du génome et les protéines localisent dans les noyaux somatiques en développement. Des expériences d’extinction de ces gènes par ARN interférence ont prouvé que ces gènes étaient indispensables. Au niveau moléculaire, l’ADN non réarrangé est amplifié dans les cellules déplétées de Ku. De plus, les cassures double brin programmées ne sont pas introduites aux bornes des IES.Mes résultats suggèrent que Ku fait partie d’un complexe de pré-excision, avec la transposase domestiquée Pgm, et est nécessaire pour l’introduction des cassures double brin programmées pendant les réarrangements programmés du génome. / Programmed elimination of germline specific DNA has been described in several eukaryotic organisms. These rearrangements are initiated through introduction of DNA double strand breaks (DSB). To ensure genome integrity, efficient repair is needed. In Paramecium tetraurelia, the genome is widely rearranged during development of a new somatic nucleus after introduction of tens of thousands of DSBs by the domesticated transposase PiggyMac (Pgm)These rearrangements consist in: the precise excision of thousands of unique and non coding sequences called IESs that interrupt 47% of genes in the germline; and the heterogeneous elimination of repeated sequences. It leads to internal deletions of variable sizes or to chromosome fragmentation with telomere addition at DNA ends.Implication of the Non Homologous End Joining Pathway (NHEJ) in precise IES excision has been proved. In cells depleted for Ligase IV or XRCC4, DSBs at IES boundaries are introduced normally but broken DNA ends accumulate without being repaired nor degraded. The repair pathway implicated in heterogeneous rearrangements is still unknown. An hypothesis would be that heterogeneous rearrangements involve a Ku independent alternative NHEJ (alt-NHEJ) pathway characterized by end resection and use of microhomologies. During my thesis I studied the role of Ku proteins in programmed genome rearrangements.Two KU70 genes and three KU80 genes has been identified in the Paramecium genome. KU70a and KU80c are specifically induced during programmed genome rearrangements. Encoded proteins localize in developing somatic nuclei. Gene extinction by RNA interference experiments proved that these genes are necessary for programmed genome rearrangements. At molecular level, non rearranged DNA is amplified in cells depleted for Ku. And more surprisingly, no programmed DSBs are introduced at IES boundaries in these cells.My results indicate that Ku is a part of a pre excision complex with the domesticated transposase Pgm and necessary for the introduction of programmed DSB during programmed genome rearrangements.

L'Efficacité Biologique Relative (EBR) des faisceaux de protons énergétiques (70-250 MeV) utilisés dans les différents centres de protonthérapie est classiquement estimée à 1,10 par rapport aux photons du Cobalt-60. Bien qu'en accord avec la mesure de la régénération des cryptes intestinales chez la souris après exposition à une dose unique de 10 à 15 Gy, cette valeur moyenne a été contestée par la microdosimétrie. Ces incertitudes nous ont conduits à analyser l'effet des protons mis en œuvre dans les deux faisceaux médicaux (76 et 201 MeV) du Centre de Protonthérapie de l’Institut Curie à Orsay (ICPO) sur deux lignées cellulaires humaines tumorales, et en partie sur une lignée fibroblastique. Les résultats font apparaître une différence de la valeur de l'EBR pour la survie au rayonnement dans la partie distale du SOBP (Spread-Out Bragg Peak) en fonction de l'énergie incidente, ainsi qu'une absence de corrélation entre la réponse en survie et l'incidence des cassures double-brin de l'ADN dans le faisceau de 76 MeV. Nous montrons cependant, grâce à l'utilisation de lignées défectives dans les voies de signalisation et de réparation des cassures double-brin de l'ADN par le D-NHEJ, que ces voies déterminent la valeur de l'EBR dans la partie distale du SOBP de 76 MeV. La réponse aux dommages de l'ADN dans cette région suggère que les dommages létaux appartiennent à la classe des “lésions complexes” (LMDS) de l'ADN. D'autre part, il apparaît que la fluence des particules constitue un paramètre majeur qui doit être pris en compte dans la partie distale des faisceaux. / Treatment planning in proton therapy uses a generic value for the Relative Biological Efficiency (RBE) of 1.1 relative to 60Co gamma-rays throughout the Spread Out Bragg Peak (SOBP). We have studied the variation of the RBE at three positions in the SOBP of the 76 and 201 MeV proton beams used for cancer treatment at the Institut Curie Proton Therapy in Orsay (ICPO) in two human tumor cell lines using clonogenic cell death and the incidence of DNA double-strand breaks (DSB) as measured by pulse-field gel electrophoresis without and with endonuclease treatment to reveal clustered lesions as endpoints.The RBE for induced cell killing by the 76 MeV beam increased with depth in the SOBP. However for the 201 MeV protons it was close to that for 137Cs gamma-rays and did not vary significantly. The incidence of DSBs and clustered lesions was higher for protons than for 137Cs g-rays, but did not depend on the proton energy or the position in the SOBP.In the second part of our work, we have shown using cell clones made deficient for known repair genes by stable or transient shRNA transfection, that the D-NHEJ pathway determine the response to protons. The response of DNA damages created in the distal part of the 76 MeV SOBP suggests that those damages belong to the class of DNA "complex lesions" (LMDS). It also appears that the particle fluence is a major determinant of the outcome of treatment in the distal part of the SOBP.

Au cours de leur développement, les cellules du système hématopoïétique sont très exposées aux dommages à l’ADN qui peuvent avoir une origine exogène ou endogène. Les organismes vivants ont développé de nombreux mécanismes de réparation pour y faire face, et leur dysfonctionnement est responsable de maladies rares mais sévères chez l’Homme. Un des deux mécanismes de réparation des cassures double-brin (CDB) de l’ADN joue un rôle prépondérant dans le développement du système immunitaire (SI) des mammifères. Il s’agit de la voie de réparation des extrémités non-homologues (NHEJ) qui est absolument essentiel au bon déroulement de la recombinaison V(D)J dans les progéniteurs lymphocytaires de la moelle osseuse et du thymus. En effet, la formation de CDB de l’ADN est une étape clé de ce remaniement. De même, bien que dans une moindre mesure, le NHEJ intervient pour réparer les cassures induites par AID lors de la commutation de classe des immunoglobulines (Ig- CSR). Notre équipe a précédemment identifié un nouveau facteur du NHEJ, Cernunnos (ou XLF), responsable chez l’Homme de déficit immunitaire combiné sévère (DCIS) associé à une sensibilité aux rayonnements ionisants (RI) et à une microcéphalie. Afin de mieux comprendre le rôle de Cernunnos dans le système hématopoïétique et dans le développement des lymphocytes en particulier, nous avons créé un modèle murin invalidé pour ce gène. De façon surprenante, le développement lymphocytaire se fait quasi normalement dans ces souris, le seul défaut observé est une diminution du nombre de lymphocytes. Cependant, l’analyse fine du répertoire des cellules T a permis de mettre en évidence un biais dans l’utilisation des segments variables V et J de la chaîne α du récepteur (TCRα). Ce serait là la signature d’un défaut de survie des thymocytes, passant par une activation chronique de la voie de l’apoptose dépendante de p53 en réponse à l’accumulation de dommages de l’ADN. Certaines sous- populations de lymphocytes T, comme les iNKTs et les MAITs, seraient ainsi affectées. Par ailleurs, notre équipe poursuit la caractérisation génétique et fonctionnelle de pathologies chez des patients dont le tableau clinique laisse penser qu’il existe un déficit immunitaire ou hématologique primaire associé à un défaut de réparation de l’ADN. Nous nous sommes intéressés à un patient dont le tableau clinique combinant déficit hématopoïétique et instabilité génomique suggère une origine génétique forte. Grâce aux techniques de séquençage haut- débit et à l’étude de ségrégation au sein de la famille nous avons pu isoler plusieurs mutations dont une nous a interpellé plus particulièrement / Throughout their development, hematopoietic cells are exposed to many DNA damages of either exogenous or endogenous origin. Living organisms evolved a variety of DNA repair mechanisms in order to face those threats, and their impairment leads to rare but severe diseases in human. Of the two mechanisms involved in the repair of DNA double-strand break (DSB) repair, one plays a major role in mammal’s Immune System (IS). The non-homologous end joining (NHEJ) pathway is essential for the correct proceeding of V(D)J recombination in lymphocyte progenitors from bone marrow and thymus. Indeed, the formation of DNA DSB is a key step of the rearrangement. In similar fashion, though to a lesser degree, NHEJ is involved in repair of AID induced breaks during immunoglobulin class switch recombination (Ig-CSR). Our team previously identified a new NHEJ factor, Cernunnos (or XLF), as being responsible for a human syndrome of severe combined immunodeficiency (SCID) associated with ionizing radiation (IR) sensitivity (RS-SCID) and microcephaly. To better understand Cernunnos role in the hematopoietic system and particularly in lymphocyte development, we engineered a knock-out (KO) mouse model for this gene. Surprisingly, lymphocyte development is almost normal in these mice, the only defect observed being a decrease of lymphocyte number. However, a refined analysis of T cell repertoire allowed us to uncover a bias in the use of V and J segments from the receptor’s α chain (TCRα). This is the signature of a survival defect in thymocytes, caused by chronic activation of the p53 dependent apoptosis pathway in response to DNA damage. Some discrete T cell populations, such as iNKTs and MAITS, would be affected. In the meantime, our team pursues the uncovering of genetic diseases and their functional description in patients showing signs of immune or hematopoietic deficiency combined to impaired DNA repair. We focused on a patient harboring clinical signs of genomic instability and hematopoietic defects with strong evidence for genetic cause. Thanks to high-throughput DNA sequencing technology and whole genome association study (WGAS), we identified several mutations, one of them striking us as pertinent

Les défauts dans la réparation des cassures double-brin de l'ADN (DSBs) peuvent avoir d'importantes conséquences pouvant entrainer une instabilité génomique et conduire à la mort cellulaire ou au développement de cancers. Dans la plupart des cellules mammifères, le mécanisme de Jonction des Extrémités Non Homologues (NHEJ) est le principal mécanisme de réparation des DSBs. L'ADN Ligase IV (LigIV) est une protéine unique dans sa capacité à promouvoir la NHEJ classique. Elle s'associe avec deux autres protéines structuralement similaires, XRCC4 et XLF (ou Cernunnos). LigIV interagit directement avec XRCC4 pour former un complexe stable, tandis que l'interaction entre XLF et ce complexe est médiée par XRCC4. XLF stimule fortement l'activité de ligation du complexe LigIV/XRCC4 par un mécanisme encore indéterminé. Récemment, un rôle structurel non catalytique a été attribué à LigIV (Cottarel et al., 2013). Dans le travail de thèse présenté ici, nous avons reconstitué l'étape de ligation de la NHEJ en utilisant des protéines recombinantes produites dans des bactéries afin d’une part, d'explorer les bases moléculaires du rôle structural de LigIV, d’autre part de comprendre le mécanisme par lequel XLF stimule le complexe de ligation, et enfin de mieux comprendre comment ces trois protéines coopèrent au cours de la NHEJ. Nos analyses biochimiques suggèrent que XLF via son interaction avec XRCC4 lié à LigIV, pourrait induire un changement conformationnel dans la LigIV. Ce réarrangement de la ligase exposerait son interface de liaison à l'ADN ce qui lui permettrait alors de ponter deux molécules indépendantes d'ADN, une capacité indépendante de l'activité catalytique de LigIV. / Failure to repair DNA double-strand breaks (DSBs) may have deleterious consequences inducing genomic instability and even cell death. In most mammalian cells, Non-Homologous End Joining (NHEJ) is a prominent DSB repair pathway. DNA ligase IV (LigIV) is unique in its ability to promote classical NHEJ. It associates with two structurally related proteins called XRCC4 and XLF (aka Cernunnos). LigIV directly interacts with XRCC4 forming a stable complex while the XLF interaction with this complex is mediated by XRCC4. XLF strongly stimulates the ligation activity of the LigIV/XRCC4 complex by an unknown mechanism. Recently, a structural noncatalytic role of LigIV has been uncovered (Cottarel et al., 2013). Here, we have reconstituted the end joining ligation step using recombinant proteins produced in bacteria to explore not only the molecular basis for the structural role of LigIV, but also to understand the mechanism by which XLF stimulates the ligation complex, and how these three proteins work together during NHEJ. Our biochemical analysis suggests that XLF, through interactions with LigIV/XRCC4 complex, could induce a conformational change in LigIV. Rearrangement of the LigIV would expose its DNA binding interface that is able to bridge two independent DNA molecules. This bridging ability is fully independent of LigIV’s catalytic activity. We have mutated this interface in order to attempt to disrupt the newly identified DNA bridging ability. In vitro analysis of this LigIV mutant will be presented as well as a preliminary in vivo analysis.

<p>Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. </p><p>The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes <i>in vivo</i> and the repair was independent of DNA-PK, XRCC1 and PARP-1.</p><p>Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G₁ phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells.</p><p>DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.</p>

Efficient and correct repair of DNA damage, especially DNA double-strand breaks (DSBs), is vital for the survival of individual cells and organisms. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. The repair of DSBs in cell lines with different DSB rejoining capabilities was studied after exposure to ionising radiation. A new cell lysis protocol performed at 0ºC, which prevents the inclusion of non-true DSBs in the quantification of DSBs by pulsed-field gel electrophoresis (PFGE), was developed. Results showed that when the standard protocol at 50ºC was used, 30-40% of the initial yield of DSBs corresponds to artifactual DSBs. The lesions transformed to DSBs during incubation at 50ºC were repaired within 60-90 minutes in vivo and the repair was independent of DNA-PK, XRCC1 and PARP-1. Non-homologous end-joining (NHEJ) is the major DSB repair pathway in mammalian cells. We show that DSBs are processed into long single-stranded DNA (ssDNA) ends after ≥1 h of repair in NHEJ deficient cells. The ssDNA was formed outside of the G1 phase of the cell cycle and only in the absence of the NHEJ proteins DNA-PK and DNA Ligase IV/XRCC4. The generation of ssDNA had great influence on the quantification of DSBs by PFGE. The standard protocol caused hybridisation of the ssDNA ends, resulting in overestimation of the DSB repair capability in NHEJ deficient cells. DSBs were also quantified by detection of phosphorylated H2AX (γ-H2AX) foci. A large number of γ-H2AX foci still remaining after 21 h of repair in an NHEJ deficient cell line confirmed the low repair capability determined by PFGE. Furthermore, in normal cells difficulty in repairing clustered breaks was observed as a large fraction of γ-H2AX foci remaining 24 h after irradiation with high-LET ions.