Les cassures Double-brin de l'ADN (DSBs) sont une menace majeure pour la stabilité du génome. Afin de se protéger des effets délétères de ces dommages, les cellules activent une voie de réponse aux cassures double-brins (DDR) qui comprend des évènements qui conduisent à la reconnaissance et à la réparation de ces cassures ainsi qu'à un délai du cycle cellulaire. Cette DDR repose largement sur 2 membres de la famille des PI3K-like kinase, ataxia telangiectasia mutated (ATM) et DNA Protein Kinase (DNAPK) dont les fonctions respectives lors de la réparation restent controversées. Grâce à l'utilisation d'une lignée cellulaire contenant l'enzyme de restriction AsiSI combinée à de la cartographie par ChIP-chip, de l'analyse de la réparation de cassures séquence-spécifique ainsi qu'à de la microscopie haute résolution, j'ai pu, au cours de ma thèse mettre en évidence que aussi bien ATM que DNAPK sont recrutées sur une région confinée autour des DSBs. Cependant, une fois recrutées, elles présentent des fonctions non-redondantes que ce soit pour la ligation des cassures ou pour l'établissement des domaines yH2AX. Concernant la réparation, DNAPK est absolument requise pour la ligation des extrémités de la cassure alors que ATM est dispensable mais promeut la fidélité. En revanche, ATM est la principale kinase requise pour l'établissement des domaines yH2AX et ce quelque soit la cassure. J'ai aussi pu mettre en évidence le fait que plusieurs cassures induites par AsiSI sont capables de se regrouper au sein d'un "foyer de réparation" et ce de manière dépendante d'ATM et indépendante de DNAPK. Cette étude éclaircit les rôles respectifs des kinases ATM et DNAPK que ce soit pour la ligation des extrémités ou l'établissement des domaines yH2AX. Enfin elle a permis de mettre en évidence un nouveau rôle d'ATM dans l'organisation spatiale de la réparation et plus précisemment dans le regroupement de plusieurs DSBs au sein de "foyers de réparation" afin d'être réparées. / DNA Double Strand Breaks (DSBs) form a major threat to the genome stability. To circumvent the deleterious effects of DSBs, cells activate the DNA damage response (DDR), which comprises events that lead to detection and repair of these lesions, as well as a delay in cell cycle progression. This DDR largely rely on two members of the PI3K-like kinase family : ataxia telangiectasia mutated (ATM) and DNA Protein Kinase (DNAPK), whose respective functions during the DDR remains controversial. Using a cell line, expressing the AsiSI restriction enzyme, combined with high resolution ChIP-chip mapping, sequence-specific DSB repair kinetics analysis and advanced high resolution microscopy, we uncovered that both ATM and DNA-PK are recruited to a confined region surrounding DSBs. However, once present at the DSB site, they exhibit non-overlapping functions on end-joining and yH2AX domain establishment. At the repair level, DNAPK is absolutely required for end-joining while ATM is dispensable although promoting repair fidelity. By contrast, ATM is the main kinase required for the establishment of the histone mark yH2AX at all breaks. We also clearly demonstrated that multiple AsiSI-induced DSBs are able to associate within "repair foci", in a manner that strictly depends on ATM, but not DNAPK, activity. Our study shed light on the respective roles of ATM and DNAPK regarding end joining and yH2AX domain establishment. Lastly it allowed us to uncover a function of ATM in the spatial organisation of the repair, more precisely in the clustering of multiple breaks within "repair foci" in order to be repaired.

The amino acid proline is unique – within a polypeptide chain, proline adopts either a cis or trans peptide bond conformation while all other amino acids are sterically bound primarily in the trans configuration. In proteins, the isomeric state of a single proline can have dramatic consequences on structure and function. Consequently, cis-trans interconversion confers both barrier and opportunity – on one hand, isomerization is a rate limiting step in de novo protein folding and on the other can be utilized as a post-translational regulatory switch. Peptidyl-prolyl isomerases (PPIs) are a ubiquitous superfamily that catalyzes the interconversion between conformers. Although pervasive, the functions and substrates of most PPIs are unknown. The two largest subfamilies, FKBPs and cyclophilins, are the intracellular receptors of clinically relevant immunosuppressant drugs that also show promise in the treatment of neurodegenerative disorders and cancer. Therefore, narrowing the knowledge gap has significant potential to benefit human health. FKBP25 is a high-affinity binder of the PPI inhibitor rapamycin and is one of few nuclear-localized isomerases. While it has been shown to bind DNA and associate with chromatin, its function has remained largely uncharacterized. I hypothesized that FKBP25 targets prolines in nuclear proteins to regulate chromatin-templated processes. To explore this, I performed high-throughput transcriptomic and proteomic studies followed by detailed molecular characterizations of FKBP25’s function. Here, I discover that FKBP25 is a multifunctional protein required for the maintenance of genomic stability. In Chapter 2, I characterize the unique N-terminal Basic Tilted Helical Bundle (BTHB) domain of FKBP25 as a novel dsRNA binding module that recruits FKBP25’s prolyl isomerase activity to pre-ribosomal particles in the nucleolus. In Chapter 3, I show for the first time that FKBP25 associates with the mitotic spindle apparatus and acts to stabilize the microtubule cytoskeleton. In this chapter, I also present evidence that this function influences the stress response, cell cycle, and chromosomal stability. Additionally, I characterize the regulation of FKBP25’s localization and nucleic acid binding activity throughout the cell cycle. Finally, in Chapter 4, I uncover a role for FKBP25 in the repair of DNA double-stranded breaks. Importantly, this function requires FKBP25’s catalytic activity, identifying for the first time a functional requirement for cis-trans prolyl isomerization by FKBP25. Collectively, this work identifies FBKP25 as a multifunctional protein that is required for the maintenance of genomic stability. The knowledge gained contributes to the exploration of PPIs as important drug targets. / Graduate

Deinococcus radiodurans est une bactérie à gram-positive connue pour son extrême résistance à une grande variété d'agents endommageant l'ADN. Parmi ces derniers, les rayonnements ionisants et la dessiccation sont les plus nocifs pour la cellule, car ils introduisent des cassures dans le génome. Les cassures double brin (CDB) sont particulièrement dangereuses et doivent être réparées de façon très efficace, afin d'éviter l'apparition de mutations pouvant mener à la mort de la cellule ou de l'organisme. La recombinaison homologue (RH) est le mécanisme le plus efficace pour la réparation des CDBs. D. radiodurans est capable de restaurer entièrement son génome en à peine 3 heures, et elle accomplit la totalité du processus par la voie RecFOR. Afin d'être réparées, les CDBs doivent d'abord être reconnu. Cette étape importante, qui a lieu peu de temps après l'apparition du dommage dans la cellule, implique la protéine RecN. RecN est recrutée dès les premières étapes de la réparation de l'ADN et des études in vivo ont démontré qu'elle avait tendance à se localiser dans des foyers discrets. Des études in vitro suggèrent également que RecN favorise l'assemblage de fragments d'ADN, une fonction décrite précédemment pour les protéines SMC (telle que cohesin), qui sont structurellement similaires à RecN. De nombreuses études structurales ont été effectuées sur la protéine de type SMC, Rad50, alors qu'à présent aucune information structurale n'est disponible pour RecN. Le travail présenté ici a porté sur la caractérisation structurale de RecN et de ses domaines. Nous avons obtenu les structures cristallines de trois constructions (se chevauchant partiellement) de RecN et une étude de diffusions des rayons X aux petits angles a été effectuée sur les domaines séparés de RecN et sur la protéine entière. Les données obtenues en solution ont complété notre étude cristallographique et nous ont permis de construire un modèle atomique de la protéine entière. Des mutations ont été conçues et les protéines mutées ont été produites et utilisées pour la caractérisation de l'activité d'hydrolyse de l'ATP caractéristique de cette famille de protéines. Des études biochimiques approfondies ont été effectuées sur les différentes constructions et mutants de RecN afin de déterminer le rôle de chacun des ses domaines. Nos résultat nous ont permis de proposer un modèle qui explique comment RecN reconnaît les CDB, maintient les deux extrémités de l'ADN, et prépare l'ADN pour la réparation par les protéines RecFOR. / Deinococcus radiodurans is a Gram-positive bacterium known for its extreme resistance to a broad variety of DNA damaging agents. Among these, Ionizing Radiations and desiccation are the most harmful for the cell, since they introduce breaks in the genome. Double Strand Breaks (DSB) are particularly hazardous for the cell and they need to be repaired very efficiently, in order to avoid mutations leading to altered, if not lethal, phenotypes. Homologous Recombination (HR) is the most efficient mechanism by which DSBs are repaired. D. radiodurans is able to completely restore its genome in only 3 hours, and it accomplishes the entire process through the RecFOR pathway. In order to be repaired, DSBs first need to be recognized. The protein believed to be responsible for this important step that takes place soon after the damage occurs in the cell, is RecN. RecN is recruited at the early stages of DNA repair and in vivo studies have demonstrated its propensity to localize to discrete foci. In vitro studies also suggest that RecN possesses a DNA end-joining activity previously observed for SMC proteins (such as cohesin), which are structurally related to RecN. Several structural studies have been carried out on the SMC-like protein, Rad50, but so far no structural information is available for RecN. The work presented here focused on the structural characterization of RecN and its constitutive domains. We obtained crystal structures of three partially overlapping constructs of RecN and Small Angle X-ray Scattering was performed on the individual domains and the full-length protein. The study of RecN in solution complemented our crystallographic study and enabled us to build a reliable, atomic model of the full-length protein. Mutations were designed and the mutant RecN proteins were produced in order to characterize the ATP hydrolysis activity of RecN, which is a conserved feature of this family of proteins. Extensive biochemical studies were carried out on wild-type and mutants of both the full-length protein and the single domains, in order to determine the role and function of each of the domains. Our results led us to propose a model for how RecN might recognize DSBs, tether two broken DNA ends and prepare the DNA for subsequent repair by the RecFOR machinery.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22700号 / 医科博第115号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 齊藤 博英 / 学位規則第4条第1項該当 / Doctor of Medical 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

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24520号 / 医博第4962号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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No description available.

The dose of curative radiotherapy (RT) for cancer is commonly limited by adverse effects presenting years later. Late reacting normal tissues are, on average, more sensitive to the size of daily doses (fractions) than early reacting normal tissues and cancers. Clinical trials have shown breast cancers to be one exception to this rule, in that they are as sensitive to fraction size as the late reacting normal tissues. This has led to the adoption of hypofractionation (use of fractions >2.0 Gy) in the UK for the adjuvant therapy of women with early breast cancer. An understanding of the molecular basis of fraction size sensitivity is necessary to improve radiotherapy outcome. In this respect, it is relevant that late reacting normal tissues have lower proliferative indices than early reacting normal tissues and most cancers. Here, we test the hypothesis that tissue sensitivity to fraction size is determined by the DNA repair systems activated in response to DNA double strand breaks (DSB), and that these systems vary according to the proliferative status of the tissue. Clinical data suggest that sensitivity of epidermis to fraction size varies over a 5-week course of RT. It resembles a late reacting normal tissue in its sensitivity to fraction size in the first week of RT and loses fractionation sensitivity by weeks 4 & 5. We used this feature of human epidermis to test how fractionation sensitivity and DNA repair changed over 5 weeks of RT. Breast skin biopsies were collected 2 h after the 1st, 5th and last fractions from 30 breast cancer patients prescribed 50 Gy/25fractions/5weeks. Sections of epidermis were co-stained for Ki67, cyclin A, p21, RAD51, 53BP1 and β1-Integrin. After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per mm of basement membrane (p=0.002), of which the majority were in S/G2 phase as judged by cyclin A staining (p<0.0003). The p21 index rose from 2.8% to 87.4% (p<0.0001) after 25 fractions, indicating cell cycle arrest in the basal epidermis. By week 5, there was a 4-fold increase (p=0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, confirming an association between activation of homologous recombination (HR) and loss of tissue fractionation sensitivity. Subsequently, CHO cell lines deficient in specific DNA repair genes were used to test molecular pathways involved in sensitivity to fraction size. We irradiated AA8 (WT), irs-1SF (XRCC3-), V3-3 (DNA-PK-) and EM9 (XRCC1-) with 16 Gy gamma-rays in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA double-strand break (DSB) repair kinetics (RAD51 & 53BP1 staining) and cell cycle analysis using flow cytometry. We found that wild-type and DNA repair defective cells acquire resistance to fractionated radiotherapy by accumulation in the late S/G2 phase of the cell cycle and increased use of HR. In contrast, the irs1SF cells, defective in HR, failed to acquire radioresistance and remained equally sensitive to ionizing radiation throughout the 3-week treatment. We also demonstrated that sensitivity to fraction size is associated with functional NHEJ. It was undetectable in V3-3 cells lacking NHEJ and thereby likely relying on HR. The high fidelity of HR, which is independent of induced DNA damage levels and hence, of fraction size, may explain the low fractionation sensitivity of cells using HR to repair radiation induced DSBs. We then wanted to investigate the modifying effects of small molecule inhibitors of DNA repair on fractionation responses. To this end we tested the effects of adding selected ATM, PARP, and DNAPK inhibitors to fractionated radiotherapy in WT CHO cells. Our results showed that the ATM inhibitor had a significant radiosensitising effect when combined with fractionated RT and resulted in loss of sparing effect of fractionation in wild type CHO cells, an observation that may be clinically relevant. We also examined DNA DSB repair kinetics (RAD51 & 53BP1 foci) with these drugs in the context of fractionated IR.

DNA double-strand breaks (DSBs) are highly genotoxic lesions, which can promote chromosomal rearrangements and tumorigenesis through oncogene activation or loss of heterozygosity (LOH) at tumour suppressor loci. To identify new genes involved in DSB repair and genome stability, an S. pombe deletion library was screened for mutants which exhibited sensitivity to the DNA damaging agents bleomycin and/or MMS. 192 mutants were isolated which exhibited increased sensitivity to one or both of these agents. These mutants were further analysed in a sectoring assay and mutants sought which exhibited elevated levels of break-induced loss and rearrangement of a non-essential minichromosome. Using this approach 57 genes were identified, including all known homologous recombination (HR) and DNA damage checkpoint genes present in the library. Further, quantitative analysis of DSB repair indicated that 25 of these genes functioned to promote efficient HR repair, thus representing a comprehensive HR gene set in fission yeast. Included in this gene set are 10 genes not previously implicated in HR repair; nse5⁺, nse6⁺, ddb1⁺, cdt2⁺, alm1⁺, snz1⁺, kin1⁺, pal1⁺, SPAC31G5.18c⁺ and SPCC613.03⁺. Detailed characterisation of ddb1Δ and cdt2Δ established a role for the Ddb1-Cul4Cdt2 ubiquitin ligase complex in HR. The findings presented here support a model in which break-induced Rad3 and Ddb1-Cul4Cdt2 ubiquitin ligase-dependent Spd1 degradation promotes ribonucleotide reductase activation and nucleotide biosynthesis, which is required for post-synaptic ssDNA gap filling during HR repair. Lastly, the role of HR genes in suppressing chromosome loss and rearrangements was examined. A striking inverse correlation between levels of gene conversion and levels of both chromosome loss and LOH was observed across the HR gene deletion set. These findings support a common and likely evolutionarily conserved role for HR genes in suppressing both chromosome loss and break-induced chromosomal rearrangements resulting from extensive end processing associated with failed HR repair.