DNA provides the instructions and regulation of cell growth and survival. Mutations in DNA can cause uncontrolled and unregulated cell proliferation, resulting in cancer. Treatment of cancer involves physical removal of these cells through surgery or inducing cell death by causing irreversible damage to DNA through chemotherapy and radiotherapy. However, natural DNA repair mechanisms may interfere with therapy and may even be increased in cases of therapy resistant cancer. The use of chemotherapy and radiotherapy leads to increased recruitment of DNA repair proteins while aggressive, therapy resistant cancers show overexpression of DNA repair proteins. Rad51 is a protein involved in the homologous recombination (HR) DNA repair process. Rad51 is recruited to sites of DNA damage caused by double stranded breaks, often generated by chemotherapy and radiotherapy. It is expected that inhibition of Rad51 will impair the HR repair process while enhancing the effectiveness of chemotherapy and radiotherapy compared to conventional means. As a result, this literature review aims to identify and examine the drug inhibitors of Rad51 in order to demonstrate the potential viability of this novel treatment in a variety of cancers.

Meiosis is necessary for sexual reproduction in eukaryotes. Genetic recombination between non-sister homologous chromosomes is needed in most organisms for successful completion of the first meiotic division. Proteins that function during meiotic recombination have been studied extensively in model organisms. However, less is known about the evolution of these proteins, especially among protists. We searched the genomes of diverse eukaryotes, representing all currently recognized supergroups, for 26 genes encoding proteins important for different stages of interhomolog recombination. We also performed phylogenetic analyses to determine the evolutionary relationships of gene homologs. At least 23 of the genes tested (nine that are known to function only during meiosis in model organisms) are likely to have been present in the Last Eukaryotic Common Ancestor (LECA). These genes encode products that function during: i) synaptonemal complex formation; ii) interhomolog DNA strand exchange; iii) Holliday junction resolution; and iv) sister-chromatid cohesion. These data strongly suggest that the LECA was capable of these distinct and important functions during meiosis. We also determined that several genes whose products function during both mitosis and meiosis are paralogs of genes whose products are known to function only during meiosis. Therefore, these meiotic genes likely arose by duplication events that occurred prior to the LECA. The Rad51 protein catalyzes DNA strand exchange during both mitosis and meiosis, while Dmc1 catalyzes interhomolog DNA strand exchange only during meiosis. To study the evolution of these important proteins, we performed degenerate PCR and extensive nucleotide and protein sequence database searches to obtain data from representatives of all available eukaryotic supergroups. We also performed phylogenetic analyses on the Rad51 and Dmc1 protein sequence data obtained to evaluate their utility as phylogenetic markers. We determined that evolutionary relationships of five of the six currently recognized eukaryotic supergroups are supported with Bayesian phylogenetic analyses. Using this dataset, we also identified ten amino acid residues that are highly conserved among Rad51 and Dmc1 protein sequences and, therefore, are likely to confer protein-specific functions. Due to the distributions of these residues, they are likely to have been present in the Rad51 and Dmc1 proteins of the LECA. To address an important issue with the gene inventory method of scientific inquiry, we developed a heuristic metric for determining whether apparent gene absences are due to limitations of the sequence search regimen or represent true losses of genes from genomes. We collected RNA polymerase I (Pol I), Replication Protein A (RPA), and DNA strand exchange (SE) sequence data from 47 diverse eukaryotes. We then compared the numbers of apparent absences to a single measure of protein sequence length and sequence conservation (Smith-Waterman pairwise alignment (S-W) scores) obtained by comparing yeast and human protein sequence data. Using Poisson correlation regression to analyze the Pol I and RPA subunit datasets, we confirmed that S-W scores and apparent gene absences are correlated. We also determined that genes encoding products that are critical for interhomolog SE in model organisms (Rad52, Rad51, Dmc1, Rad54, and Rdh54) have been lost frequently during eukaryotic evolution. Saccharomyces cerevisiae null rad52, dmc1, rad54, and rdh54 mutant phenotypes are suppressed by rad51 overexpression or mutation. If rad51 overexpression or mutation affects other eukaryotes in a similar fashion, this phenomenon may account for frequent losses of genes whose products are critical for the completion of meiosis in model organisms. Finally, we place this work into greater context with a review of hypotheses for the selective forces and mechanisms that resulted in the origin of meiosis. The review and the data presented in this thesis provide the basis for a model of the origin of meiotic genes in which meiosis arose from mitosis by large-scale gene duplication, following a preadaptation that served to reduce increased numbers of chromosomes (from diploid to haploid) caused by erroneous eukaryotic cell-cell fusions.

The novel method herein proposed, aims to study Deoxyribonucleic acid (DNA) and Rad51 repair protein in its resting state after their interaction by using a combination of biological preparation and physical measures. Rad51 is a highly conserved protein; it is involved in eukaryotes genome stability and can interact with single strand (ss) and double strands (ds) DNA. In our work, a droplet of the solution containing the dsDNA/Rad51 complexes was deposited on micro-fabricated super-hydrophobic substrates (SHS) to obtain self-organized and suspended fibers. The silicon-based SHS were designed to incorporate a regular circular array of pillars and to maintain a high contact angle with the drop. The samples were let dehydrate at controlled temperature and humidity conditions. At the end of the buffer evaporation process, non-suspended material and salt excess are concentrated on the top of a few micro-pillars in a limited area (drop residual) of the device while ordered and self-assembled DNA/Rad51 fibers are suspended between micro-pillars. To find the ideal conditions to obtain and suspend the nucleic acid/protein complexes, several parameters were investigated: saline buffer, DNA and protein concentrations were widely titrated and showed a significant effect on the biomolecule suspension on SHS. The samples were then preliminarily checked by microscopy techniques and then described by the Raman spectra acquired. Several techniques were used: optical microscopy, Energy Dispersive X-Ray Spectroscopy (EDAX), Scanning Electron Microscopy (SEM) and Raman Spectroscopy. Protein expressions, DNA suspension, micro-fabrication and characterization were all performed in KAUST Core Labs and Structural Molecular Imaging Light Enhanced Spectroscopies (SMILEs) Lab. The novel approach presented in this work is highly multidisciplinary and comprises physical measurements (Raman spectroscopy and EM imaging), chemistry and biology. In future the method can be used further expanded supporting the data with HRTEM direct imaging to elucidate the nucleic acids/proteins behavior in the multiple phases of the genome repair processes. Also, it and can serve as a fingerprint of the biological molecules involved in biological interactions, their localization and structural characterization, providing a new tool for structural analysis, screening and diagnostics.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20668号 / 医博第4278号 / 新制||医||1024(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 武田 俊一, 教授 岩井 一宏, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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.

La Recombinaison Homologue (RH) est une des voies majeures, hautement fidèle, de réparation des cassures double brin de l’ADN et du redémarrage des fourches de réplication arrêtées ou bloquées. La RH utilise une séquence homologue pour réparer avec précision l'ADN. Elle est essentielle pour le maintien de la stabilité des génomes dans tous les organismes et également pour assurer la transmission et l'échange de l'information génétique pendant la méiose. L'étude mécanistique de la RH est importante pour comprendre l'instabilité génétique, la perte d'hétérozygotie, les aberrations chromosomiques, la mort cellulaire et la cancérogenèse associée à une RH déficiente. Les étapes clés de la RH et les protéines impliquées sont très conservées dans toutes les espèces. Chez Saccharomyces cerevisiae, la recombinase Rad51 forme un filament présynaptique avec l’ADNsb qui est capable de rechercher les homologies de séquences dans tout le génome, en partenariat avec d'autres partenaires protéiques. Une fois l'homologie identifiée, une structure de D-loop (pour Displacement loop) est formée pour favoriser l'échange de brins. Le moteur moléculaire Rad54 assiste Rad51 dans la formation de la D-loop. Son rôle dans la recherche d'homologie et la formation des complexes synaptiques, avant mêle la formation de la D-loop reste un sujet de débat. Cette thèse porte sur mes travaux d’étude in vitro des mécanismes de formation de la D-Loop, en utilisant des protéines de la RH purifiées Rad51 et Rad54 avec d'autres partenaires protéiques et des substrats d'ADN synthétisés, mimant les structures de la RH. J'ai utilisé la microscopie électronique (ME) pour visualiser directement l'ADN et les complexes ADN-protéines intervenant au cours de la formation de D-loop in vitro avec Rad51, Rad54 et un mutant de Rad54. Ces approches d’imagerie, combinées à la biochimie suggèrent que Rad54 est crucial pour la recherche d'homologie et la formation du complexe synaptique, avant la formation de la D-loop, dans une coopération étroite avec Rad51. J'ai également montré que les paralogues de Rad51, Rad55-Rad57, stimulent la formation de la D-loop et que cet hétérodimère présente une activité ATPase dix fois plus forte que Rad51. Par ailleurs, j'ai également développé d'autres outils méthodologiques en ME et en microscopie à force atomique à haute vitesse (HS-AFM) pour mieux caractériser différents intermédiaires de la RH. / Homologous recombination (HR) is a major high-fidelity DNA repair pathway of double-stranded breaks and recovery of stalled and collapsed replication forks. HR uses a homologous template to accurately repair DNA that is essential for maintaining genomic stability in all organisms and to ensure the transmission and exchange of the genetic information during meiosis. The importance of HR study is highlighted by genetic instability, loss of heterozygosity, chromosomal aberrations, cell death and carcinogenesis associated with a defected HR. The key recombinational stages and proteins are well conserved throughout species. In Saccharomyces cerevisiae, the Rad51 recombinase forms a presynaptic filament with ssDNA that along with other protein partners is able to search for homology within the entire genome. Once homology is identified, a Displacement-loop (D-loop) is formed to promote strand-exchange. The Rad54 molecular motor assists Rad51 in the D-loop formation, and it is still a matter of debate whether it also plays a key role in homology search and synaptic complex formation, prior to D-loop. This dissertation covers my in vitro assays using purified key HR proteins Rad51 and Rad54, other protein partners and designed DNA substrates, mimicking HR structures.I used electron microscopy (EM) to directly visualize the HR DNA and DNA-protein complexes generated by D-loop in vitro assay with Rad51, Rad54 and a Rad54 mutant, and these studies combined with biochemistry suggest Rad54 is crucial to homology search and synaptic complex formation, prior to D-loop formation, in a tight intercooperation by Rad51 and Rad54. In a multiprotein system, I also showed the Rad51 paralogs Rad55-Rad57 stimulate the D-loop formation and that this heterodimer presents a ten times stronger ATPase activity than Rad51. I also developed other EM and high speed atomic force microscopy (HS-AFM) methodological tools to characterize other HR intermediates.

Les tumeurs mammaires sont connues pour présenter une grande hétérogénéité intratumorale qui contribue à l’échec thérapeutique et à la progression de la maladie. L’origine de dette hétérogénéité s’explique principalement par l’organisation hiérarchique des tissus tumoraux où plusieurs sous-populations de cellules souches de cancer du sein (bCSC) sont capables de s’auto-renouveler et de maintenir l’architecture oligoclonale de la tumeur. Dans la mesure où les bCSC stimulent la croissance tumorale, résistent aux thérapies conventionnelles et initient le développement des métastases, il est indispensable de développer des thérapies spécifiques ciblant ces cellules. L’élaboration d’une telle stratégie nécessite la compréhension des propriétés moléculaires intrinsèques des bCSC. Pour mieux comprendre leur biologie, nous avons isolé les bCSC de différentes xénogreffes dérivées de tumeurs de patientes et établit leurs profil d’expression génique. Nous avons identifié un programme transcriptionnel pouvant être impliqué dans la réduction du stress réplicatif (SR) des bCSC . Nous avons montré que comparé aux non-bCSC, les bCSC présentent une sur-activation de la recombinaison homologue qui leur permet de réduire leur niveau de SR. Nous avons ensuite montré en réalisant un essai clinique que l’inhibition de cette voie permet de les sensibiliser à des agents génotoxique. Ces travaux identifient le SR comme le talon d’Achille des bCSC et mettent en évidence la recombinaison homologue comme cible potentielle pour sensibiliser les BCSC aux thérapies conventionnelles. / Breast tumors are known to present a major intratumoral heterogeneity that contributes to therapy failure and disease progression. The origin of this cellular heterogeneity is mainly explained by a hierarchical organization of tumor tissues where several subpopulations of self-renewing breast cancer stem cells (bCSCs) sustain the long-term oligoclonal maintenance of the neoplasm. bCSCs drive tumor growth, resist to conventional therapies and initiate metastasis development. Thus, developing bCSC-targeting therapies is becoming a major challenge requiring the understanding of the unique molecular circuitry of bCSC as compared to non-bCSC. To better understand the biology of these cells, we isolated bCSCs from different patient–derived xenografts (PDXs), derived fom breast tumors, and established their gene expression profiles. We identified a bCSC core transcriptional program that may be implicated in the reduction of the replicative stress in CSC: overexpression of genes implicated in dNTP metabolism and homologous recombination (HR). Our results show that HR plays a major role in SR regulation of bCSC and that bCSC are more resistant to RS than non-bCSC, We realized a preclinical assay in PDX and showed that HR inhibition prevent bCSC expansion Cisplatin-induced, suggesting a sensitization of the bCSC to the chemotherapy. Our results identify replication stress as the Achilles’ heel of bCSC and highlights HR as potential targets for anti-bCSC therapy.

Les glioblastomes (GBMs) sont des tumeurs cérébrales au pronostic défavorable. La résistance aux thérapies actuelles et la rechute des GBMs pourraient être due à l'existence de cellules aux propriétés souches. L'objectif de ma thèse a été la caractérisation des cellules souches de GBMs (CSGs) isolées à partir de tumeurs. L'analyse du profil souche et de pluripotence des CSGs a montré qu'elles sont maintenues dans un état souche par SOX2 et que COL1A1 et IFITM1 peuvent être des cibles thérapeutiques potentielles. L'étude de la radiosensibilité des CSGs à travers l'analyse des courbes de clonogénicité a mis en évidence deux groupes dont un «atypique» pouvant être composé de sous-populations de cellules aux radiosensibilités différentes qu'il conviendra de caractériser. L'étude de la réparation a mis en évidence deux autres groupes dont un ayant un fort potentiel de réparation qui exprime plus fortement le gène RAD51 après irradiations. Le traitement par un inhibiteur spécifique de RAD51 ralentirait la capacité de réparation de ces cellules. Malgré cette hétérogénéité, l'inhibition de la voie Hedgehog (HH) par un vecteur glucuronylé de la cyclopamine, activé par le microenvironnement tumoral, inhibe la prolifération et l'auto-renouvellement des CSGs in vitro et ralentit la croissance tumorale in vivo. La voie HH semble être une cible thérapeutique intéressante commune à toutes les CSGs. Néanmoins, il est nécessaire de prendre en compte l'hétérogénéité dans les populations tumorales pour le développement de la médecine personnalisée. / Glioblastomas (GBMs) are brain tumors with a poor prognosis. Their resistance to current therapies and the occurrence of tumor relapse may be related to the existence of cells bearing stem cell characteristics. The aim of this PhD research was to characterize glioblastoma stem cells (GSCs) having been isolated from tumors. Analysis of the stemness and pluripotency profiles of GSCs indicated that their stemness states are maintained by SOX2 and that COL1A1 and IFITM1 may be potential therapeutic targets. Clonogenic studies of GSC radiosensitivity underscored the presence of two groups, one of them composed of sub-populations of cells with different degrees of radiosensitivity that have yet to be fully characterized. Study of DNA repair capacity highlighted two additional groups including one with high repair potential overexpressing the RAD51 gene after 4Gy. However, treatment with RAD51 inhibitor is likely to slow down repair of GSC lesions. Notwithstanding GSC heterogeneity, in our study inhibition of the Hedgehog pathway (HH) by a cyclopamine glucuronid prodrug, activated by the tumor microenvironment, inhibited in vitro proliferation and self-renewal in all the GSCs tested and slowed down tumor growth in vivo. Hence, HH pathway appears to be conserved among GSCs and constitutes an interesting potential therapeutic target. With regard to the development of personalized medicine, it is nevertheless highly advisable to take into account the pronounded heterogeneity of tumor populations.

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