Les cellules utilisent deux stratégies principales pour réparer les cassures double-brin (CDB) de l’ADN : la recombinaison homologue (RH) et la ligature d’extrémités non homologues (NHEJ). D’autres voies de réparation plus minoritaires existent qui mènent nécessairement à des altérations génétiques : Single Strand Annealing (SSA) et Alternative End Joining (A-EJ). Nous avons proposé que le choix entre les mécanismes de réparation des CDB nécessite deux étapes : 1) la compétition entre C-NHEJ et la résection, 2) sur les extrémités d’ADN résectées, la compétition entre RH, A-EJ et SSA. Ici, nous avons étudié la régulation de la deuxième étape de ce choix. En outre, la létalité synthétique a été décrite entre RAD52 et BRCA2/PALB2. Étant donné que BRCA2 et PALB2 sont nécessaires pour le chargement de RAD51 sur l’ADN simple brin, cela suggère que la formation d’un nucléofilament RAD51/ADNsb ordonné et RAD52 sont des acteurs essentiels dans le choix de la réparation à la deuxième étape. Nous avons trouvé que l’extinction de RAD51 ou BRCA2 stimule à la fois le SSA et EJ, d’une manière épistatique et que RAD52 contrôle la stimulation de SSA et A-EJ, en absence de RAD51. De plus, par séquençage haut débit, nous montrons que l’inhibition de RAD51 induit une instabilité génomique médiée par la microhomologie au niveau du génome. Cependant l’inhibition de RH n’est pas la réparation directe suffisante vers SSA et A-EJ. En effet, en utilisant des mutants dominants négatifs de RAD51, nous avons trouvé que les mutants du site de fixation/hydrolyse de l’ATP inhibent la RH et stimulent le SSA et que la chimère SMRAD51, qui inhibe la RH, inhibe également le SSA et EJ. Par TEM, nous avons observé que SMRAD51 perturbe spécifiquement la structure de l’ADNsb/SMRAD51. De l’autre côté, deux mutants d’hydrolyse de l’ATP de RAD51 ont montré que la liaison à l’ATP et l’hydrolyse d’ATP sont nécessaires pour une charge efficace de RAD51 sur l’ADN endommagé, dans les cellules vivantes. Ces deux mutants d’ATP ne se fixent pas à l’ADN en opposition à SMRAD51. Enfin, nous montrons que RAD51 n’empêche pas la résection étendue, mais que, in vitro, la protéine RAD51 empêche l’annealing de l’ADNsb complémentaire. Au total, les données montrent que RAD51 joue effectivement un rôle crucial dans la deuxième étape du choix de la voie de réparation des CDB à travers deux mécanismes distincts : 1- il déclenche la RH par son activité catalytique, 2- mail il empêche également les mécanismes non conservateurs dépendants de RAD52, SSA et A-EJ, en altérant l’étape de l’annealing. Par conséquent, le choix en deuxième étape entre la RH et les mécanismes mutagènes, SSA et A-EJ, est orchestré par un antagonisme entre RAD51 et RAD52. / Cells use two primary strategies to repair DNA double-strand break (DSB): Homologous Recombination (HR) and Non-homologous end joining (NHEJ). Beside other mechanisms exist that necessarily lead to genetic alterations: Single Strand Annealing (SSA) and Alternative End Joining (A-EJ). We have proposed that the choice between DSB repair mechanisms requires two steps: 1) competition between C-NHEJ and resection; 2) on resected DNA ends, competition between HR, A-EJ and SSA. Herein we investigated the regulation of the second step of this choice. Furthermore, synthetic lethality has been described between RAD52 and BRCA2/PALB2. Since BRCA2/PALB2 are required for the loading of RAD51 onto the ssDNA, suggesting that both the formation of an ordered RAD51/ssDNA nucleofilament and RAD52 are central players in the choice of repair at the 2nd step.We found that silencing RAD51 or BRCA2 stimulate both SSA and EJ, in an epistatic manner and that silencing RAD51 induced microhomology mediated genomic instability at a genome wide level. Moreover, we show that RAD52 controls the stimulation of SSA and A-EJ, upon RAD51 silencing. However inhibition of HR is not sufficient redirect repair toward SSA and A-EJ. Indeed, using dominant negative mutants of RAD51 we found that the chimera SMRAD51, which inhibits HR, also inhibits SSA and EJ. By TEM we observed that SMRAD51 specifically disrupts the structure of the ssDNA/SMRAD51. On the other side, two ATP hydrolysis mutants of RAD51 showed that ATP binding and hydrolysis is required for efficient loading of RAD51 on damaged DNA, in living cells. These two ATP mutants that do not bind DNA in opposition to SMRAD51, do not inhibit A-EJ and stimulate SSA. Finally we show RAD51 do not prevents extended resection, but that, in vitro, RAD51 protein prevents the annealing of complementary ssDNA.Altogether the data show that RAD51 indeed plays a pivotal role in the second step of DSB repair pathway choice through two separable mechanisms: 1- it triggers HR through its catalytic HR activity 2- but it also prevents RAD52-dependent non-conservative mechanisms SSA and A-EJ, by impairing the annealing step. Therefore, the choice between HR and alternative mutagenic mechanisms A-EJ and SSA (2nd step) is orchestrated by an antagonism between RAD51 and RAD52

Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair. A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis. Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA. We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.

Repair of double-strand DNA breaks (DSBs) by the homologous recombination (HR) pathway results in crossovers (COs) required for a successful first meiotic division. DSB resection is the nucleic degradation of DSB ends to expose 3’ single strand DNA (ssDNA), an intermediate required for HR. To investigate genes involved in meiosis, a forward genetic screen was performed to search for novel genes or informative new mutant alleles of known genes. Mre11 is one member of the MRX/N (Mre11-Rad50-Xrs2/Nbs1) complex required for meiotic DSB formation and for resection in budding yeast. In Caenorhabditis elegans, evidence for the MRX/N’s role in DSB resection is limited. We isolated the first separation of function allele in C. elegans , mre 11(iow1), isolated from our forward genetic screen. The mre-11(iow1) mutants are specifically defective in meiotic DSB resection but not in DSB formation. The mre 11(iow1) mutants display chromosomal fragmentation and aggregation in late prophase I. Recombination intermediates and crossover formation is greatly reduced in mre 11(iow1) mutants. Irradiation induced DSBs during meiosis fail to be repaired from the early to middle prophase I in mre 11(iow1) mutants. Our data suggest that some DSBs in mre 11(iow1) mutants are repaired by the non homologous end joining (NHEJ) pathway because removing NHEJ partially suppresses some meiotic defects conferred by mre 11(iow1). In the absence of NHEJ and a functional MRX/N, meiotic DSBs are channeled to an EXO 1 dependent form of recombination repair. Overall, our analysis supports a role for MRE-11 in the resection of DSBs in early to middle meiotic prophase I and in blocking NHEJ. A reverse genetic screen and a yeast two hybrid screen were performed to search for genes with genetic and/or physical interactions with mre-11. The reverse genetic screen isolated a novel meiotic gene, nhr-2, as a partial suppressor of the meiotic defects conferred by mre-11(iow1). The yeast two hybrid screen identified kin-18 interacting with mre-11. KIN-18 is the C. elegans homolog of mammalian Thousand And One kinase (TAO) kinase. KIN-18/TAO is MAPK kinase kinase whose meiotic role was unknown. We have found that KIN-18 is essential for normal meiotic progression as kin-18 mutants exhibit accelerated meiotic recombination, ectopic germ cell differentiation, and enhanced levels of germline apoptosis. In C.elegans MPK-1 activation in late pachytene is required for physiological apoptosis (nuclei removed by apoptosis serve as nursing cells for oocytes) and oocyte differentiation. The kin-18 mutants also showed absence of MPK-1 activation and aberrant MPK-1 activation that includes ectopic activation in the wrong regions in the germline or more than one time of activation. The progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator, KSR-2, of the canonical MPK-1 signaling. Our data suggest KIN-18 affects meiotic progression by modulating the timing of MPK-1 activation. This regulation ensures the proper timing of recombination and normal apoptosis, which is required for the formation of functional oocytes. Meiosis is a conserved process; revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans will motivate hypothesis for TAO kinase’s role in the germline development in higher eukaryotes. Meiosis is a crucial for sexually reproducing organisms to maintain ploidy level from one generation to the next. Accurate chromosome segregation in the meiosis requires meiotic recombination between homologous chromosomes. Failure in recombination can lead to abnormal segregation of chromosomes in meiosis, which leads to aneuploidy. Anueploidy is a leading cause of miscarriages and attributes to chromosomal related birth defects. Meiotic recombination starts with programmed DNA double strand breaks (DSBs), followed by repair of these DSBs by homologous recombination (HR) pathway. One key step in HR is resection, a process to covert DSB ends into single strand DNA (ssDNA). To broaden our understanding of meiotic DSB resection, we used a nematode, C. elegans, as a model to investigate genes in DSB resection. We have isolated a specific mutant allele of a meiotic gene, mre-11. Our data suggest meiotic DSB resection in C. elegans requires collaboration of mre-11 and another gene exo-1; efficient resection of DSB ends is important to safeguard repair of DSB by HR against other illegitimate repair pathway. In addition, we identified a gene kin-18 by looking for genes interacting with mre-11. Characterization of kin-18 show meiotic recombination is tightly coordinated with germ cell progression. Our analysis provides significant improvement in the understanding of meiotic recombination in C. elegans. Given the high conservation of the two genes, mre-11 and kin-18, our finding may be applied to other organisms.

Chez les mammifères, la réparation des CDBs par la voie NHEJ (Non Homologous End Joining) implique plusieurs complexes multi-protéines : (i) de reconnaissance (ADN-Ku70/Ku80), (ii) de maturation et (iii) de ligation comprenant XRCC4, XLF et ligase IV. Si les protéines impliquées dans le NHEJ sont connues, leurs propriétés structurales et fonctionnelles le sont moins. Au cours de ma thèse, j’ai combiné des approches biochimiques, de Microscopies Electronique et à Force Atomique, pour caractériser les propriétés de XRCC4, de XLF et leurs interactions avec le complexe de reconnaissance en particulier Ku. J’ai montré que la protéine complète XRCC4 est capable de polymériser et former des filaments alors qu’elle ne peut pas en faire en absence de la région C-terminale. Par Microscopies Electronique et à Force Atomique; nous avons montré que le filament XRCC4 forme une structure hélicoidale de chiralité gauche. XLF seule ne forme pas de filament mais peut être incorporé dans le filament XRCC4, ce que nous avons montré par immunomarquage. L’analyse d’images réalisé en collaboration et avec l’algorithme d’Edward Egelman (Université de Virginie-USA) a permis d’obtenir une reconstruction 3 D du filament XRCC4. Il est composé de 2 filaments enroulés l’un autour de l'autre avec un pas de 54 nm. Des Etudes sont en cours afin d’obtenir une structure 3D en CryoEM à haute résolution. L’étude des propriétés physicochimiques de l’assemblage du filament en fonction de la concentration, la température et le temps d’incubation a permis de montrer la dynamique du filament avec une stabilisation à basse température, et une concentration située entre 50 et 250 nM. L’ADN avec ou sans extrémités interagit avec les filaments. Cette interaction stabilise et promeut l’extension du filament. L’incorporation de XLF stabilise aussi le filament XRCC4. L’analyse des complexes formés entre l’ADN et XRCC4 ou XLF à l’état oligomérique montre des événements de pontage intra ou intermoléculaires. Parallèlement, nous avons étudié les propriétés de reconnaissance de l’ADN par l’hétérodimère Ku70/Ku80 et avons montré que le domaine KBM de XLF interagit avec Ku80 au sein de l’hétérodimère. En conclusion, nous montrons que le filament XRCC4 pourrait jouer un rôle d’architecture en maintenant les extrémités physiquement proches. Le recrutement de XLF dans le filament XRCC4 permettrait d’assembler le complexe de ligation avec le complexe de reconnaissance grâce aux interactions entre Ku et XLF. / In mammals, DSBs repair by the Non Homologous End Joining (NHEJ) pathway involves several multi-protein complexes : (i) the recognition complex (the Ku70 / Ku80 heterodimer), (ii) the maturation complex and (iii) the ligation complex comprising XRCC4, XLF, PAXX and ligase IV. If the proteins involved in NHEJ are identified and characterized, their structural and functional properties are often poorly understood. During my thesis I combined biochemical approaches, and molecular microscopies (Electron Microscopy and Atomic Force Microscopy), to characterize the properties of XRCC4, XLF and their interactions with the recognition complex (Ku-DNA). I have shown that the full length XRCC4 forms oligomers in solution (dimers and tetramers) and it polymerize into filaments whereas it can’t do it when the C-terminal region is absent. We initially characterized the structure of this filament in Electron Microscopy and Atomic Force Microscopy. XRCC4 filament forms a helicoidal structure of left chirality. XLF alone does not forms a filament but can be incorporated into the XRCC4 filament, which we have shown by immunostaining with gold beads. In collaboration with Edward Egelman (University of Virginia-USA), the image analysis performed using his algorithm allowed us to obtain a 3D reconstruction of the XRCC4 filament. It consists of 2 filaments wound around each other in a helical manner with a pitch of 54 nm. Studies in CryoEM are in progress to obtain a 3D high resolution structure. The study of the physicochemical properties of the filament assembly as a function of the concentration, the temperature and the incubation time allowed to show the dynamics of the filament with stabilization at low temperature, and a concentration between 50 and 250 nM. DNA with or without ends interacts with the filaments. This interaction stabilizes and promotes the extension of the filament. Similarly, incorporation of XLF stabilizes the Xrcc4 filament. Analysis of complexes formed between DNA and XRCC4 or XLF in the oligomeric state shows intra- or intermolecular bridging events. In parallel, we have studied the DNA recognition properties of the Ku70/Ku80 heterodimer and we have shown that KBM domain of XLF interacts with Ku80 within the heterodimer. In conclusion, we show that the XRCC4 filament could play an architectural role favoring repair events by keeping the ends physically close. The recruitment of XLF into XRCC4 filaments ans its interaction with Ku allow the link between ligation and recognition complexes.

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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24521号 / 医博第4963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 斎藤 通紀, 教授 萩原 正敏, 教授 戸井 雅和 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

DNA double strand breaks (DSBs), which may occur during DNA replication or due to the action of genotoxic agents, are extremely dangerous DNA lesions as they can cause chromosomal rearrangements and cell death. Therefore, accurate DSB repair is vital for genome stability and cell survival. Two main mechanisms serve to repair DNA DSBs: non-homologous end joining, which re-ligates DNA ends together, and homologous recombination (HR), which restores broken DNA using homologous sequence as a template for repair. One-ended DSBs are a subject for the specialised HR-dependent repair pathway known as break-induced replication (BIR). At low frequency, DNA breaks can also be healed by telomerase, which normally extends telomeres at natural chromosome ends, but may also add de novo telomeres to DSBs due to their similarity to chromosome ends. De novo telomere addition is a deleterious event, which is effectively inhibited by the nuclear Pif1 (nPif1) helicase phosphorylated at the TLSSAES motif in response to DNA damage. In this study, it is reported that the same regulatory motif of nPif1 is also required for DSB repair via BIR. The requirement of the nPif1 TLSSAES sequence in BIR is dependent on the functional DNA damage response (DDR). Thus, nPif1 phosphorylation by the DDR machinery might mediate the role of nPif1 in BIR. In contrast, the nPif1 regulatory motif is not essential for BIR at telomeres in cells lacking telomerase. These observations indicate that the mechanism of nPif1 function in DSB repair via BIR and in BIR at telomeres might be different. In this work, a protocol for nPif1 pull-down was optimized to reveal the mechanism of the phosphorylation-dependent nPif1 functions in cells undergoing DNA repair, i. e. the mechanism of nPif1-mediated inhibition of de novo telomere addition and promoting DSB repair via BIR. In future, this protocol can be used to dissect the role of nPif1 in DNA repair through the identification of its potential interacting partners. The Srs2 helicase negatively regulates HR via dismantling Rad51 filaments. According to preliminary data from the laboratory of Sveta Makovets, Srs2 also promotes de novo telomere addition at DSBs in a Rad51-dependent manner. The work presented here establishes that Srs2 is dispensable for telomerase-mediated addition of TG1-3 repeats to DSBs. Instead, Srs2 is required for the reconstitution of the complementary DNA strand after telomerase action, thus ensuring the completion of de novo telomere addition. Overall, this study demonstrates that recombination-dependent DSB repair and de novo telomere addition share common regulatory components, i. e. the nPif1 helicase phosphorylated in response to DNA damage and the Srs2 helicase. Phosphorylated nPif1 promotes DSB repair via BIR in addition to its known role in inhibition of telomerase at DSBs, whereas Srs2 uses its well established ability to remove Rad51 from ssDNA to promote the restoration of dsDNA and thus to complete de novo telomere addition.

La surexpression de la lamine B1, un composant majeur de l'enveloppe nucléaire, a été rapportée dans diverses tumeurs. Cependant, les causes et les conséquences de cette augmentation sur la stabilité du génome n'ont pas été étudiées à ce jour. En effet, l'instabilité du génome est considérée comme une caractéristique majeure des cellules cancéreuses. Pour assurer le maintien de la stabilité du génome, les cellules ont développé de multiples et complexes mécanismes parmi lesquels les voies de réparation de l'ADN et la gestion du stress réplicatif sont essentielles. Au cours de ma thèse, l'impact de l'augmentation de niveau de lamine B1 sur la stabilité du génome, en particulier sur la réparation de cassure double-brin (CDB) et sur le contrôle du stress réplicatif a été étudié. En effet, nous montrons qu'une augmentation de la lamine B1 entraîne une accumulation de CDB et leur persistance en réponse à l'irradiation (foyers γH2AX), en plus d'une sensibilité accrue à l'irradiation (formation de colonies et cassures chromosomiques). Les cellules surexprimant la lamine B1 montrent également des défauts de recrutement de 53BP1 aux sites de dommages d’ADN, couplés à une diminution de l'efficacité de la réparation de CDB par NHEJ (Non-Homologous End-Joining). De plus, nous avons identifié une interaction directe entre la lamine B1 et 53BP1 régulant le recrutement de ce dernier aux CDB. Nos résultats supportent un modèle dans lequel l'augmentation de la lamine B1 conduit à la séquestration de 53BP1, modifiant ainsi son recrutement aux CDBs. En parallèle, nous montrons que les cellules surexprimant la lamine B1 présentent des signes accrus de stress réplicatif tels que l'accumulation de foyers spontanés de p-RPA, l'augmentation des figures radiales lors du traitement par mitomycine C, et une sensibilité accrue au traitement par camptothécine. Nous avons en outre cherché à identifier les causes de l'augmentation du stress réplicatif dans ces cellules, et les conséquences potentielles, en particulier sur l'induction de phénotypes inflammatoires. En fait, nous montrons que la surexpression de la lamine B1 conduit à une diminution de l'efficacité de la réparation de CDB par la recombinaison homologue, couplée à un défaut de formation de foyers BRCA1 après irradiation. De plus, nous avons obtenu des données préliminaires suggérant une induction de l'inflammation lors de la surexpression de la lamine B1. En résumé, ce travail de Thèse a permis d’identifier un nouveau mécanisme régulant le recrutement de 53BP1 aux CDB par son interaction avec la lamine B1, et souligne le rôle de l'augmentation de la lamine B1 dans la promotion de l'instabilité génomique au moins partiellement par des défauts de réparation de CDB et une augmentation de stress réplicatif. Après confirmation de l'induction de phénotypes inflammatoires, nous aurions identifié des rôles de l'augmentation de la lamine B1 dans la promotion de deux caractéristiques majeures du cancer - l'instabilité génomique et l'inflammation - favorisant ainsi le rôle de la lamine B1 dans le développement tumoral et proposant cette dernière comme une cible thérapeutique antitumorale potentielle. / The overexpression of lamin B1, a major component of nuclear envelope, has been reported in various tumors. However, the causes and consequences of this increase on the genome stability have not been studied to date. Indeed, genome instability is considered a major hallmark of cancer cells. To ensure the maintenance of genome stability, cells have developed multiple complex mechanisms among which pathways of DNA repair and replication stress management are essential. Therefore, during my thesis the impact of an increased lamin B1 level on genome stability, in particular on double-strand break (DSB) repair and on the control of replication stress was studied. Indeed, we show that increased lamin B1 leads to an accumulation of DSBs and their persistence in response to irradiation (γH2AX foci), in addition to an increased sensitivity to irradiation (colony formation and chromosomal breaks). Lamin B1 overexpressing cells also show defects in the recruitment of 53BP1 to damage sites, coupled to a decreased efficiency of DSB repair by Non-Homologous End-Joining. Moreover, we identified a direct interaction between lamin B1 and 53BP1 regulating the latter’s recruitment to DSBs. Our results support a model where increased lamin B1 leads to the sequestration of 53BP1, thereby altering its recruitment to DSBs. In parallel, we show that cells overexpressing lamin B1 display increased signs of replication stress such as accumulation of spontaneous p-RPA foci, increased radial chromosomes upon mitomycin C treatment, and enhanced sensitivity to treatment with camptothecin. We further aimed to identify the causes of the increased replication stress in these cells, in addition to the potential consequences, in particular on the induction of inflammatory phenotypes. In fact, we show that lamin B1 overexpression leads to a decreased efficiency of DSB repair by Homologous Recombination, coupled to a defect in irradiation-induced BRCA1 foci formation. In addition, we obtained preliminary data suggesting a possible induction of inflammation upon lamin B1 overexpression. Altogether, this work identifies a novel mechanism regulating the recruitment of 53BP1 to damage sites through its interaction with lamin B1, and highlights the role of increased lamin B1 in promoting genome instability at least partially through defective DSB repair and increased replication stress. Upon confirming the induction of inflammatory phenotypes, we would have identified roles of increased lamin B1 in promoting two major hallmarks of cancer – genomic instability and inflammation - thereby favorizing a role for lamin B1 in tumor development and proposing the latter as a potential anti-tumor therapeutic target.

Genetic information must be protected, maintained and copied from cell to daughter cells, from generation to generation. In plants, most of the cells contain complete genetic information, and many of these cells can regenerate to a whole new plant. Such a feature leads to the need for precise control of which genes will be active and which not because in growth and differentiation, only the activity of specific genes for the individual cells, tissues, organs are required. One of the mechanisms controlling the gene activity is RNA interference (RNAi), which down- regulates or blocks the expression of specific genes at the transcriptional or post-transcriptional level. The crucial part of the RNAi is guiding the RNAi machinery to the target. It is mediated via sequence complementarity of the target with a small RNA (sRNA), which is diced from a double- stranded RNA (dsRNA) precursor. The molecular mechanism of dsRNA and sRNA formation and also the target origin predestinates the subsequent silencing pathway. In transcriptional gene silencing (TGS), the gene expression is regulated through chromatin epigenetic modifications. One of the epigenetic marks is cytosine methylation, which is established mainly by RNA-directed DNA-methylation (RdDM) pathway. Although the protein machinery was relatively...

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB) are among the most lethal forms of DNA damage and can occur as a result of ionizing radiation (IR), radiomimetic agents, endogenous DNA-damaging agents, etc. If left unrepaired DSB’s can cause cell death, chromosome translocation and carcinogenesis. In humans, DSB are repaired predominantly by the non-homologous end joining (NHEJ) pathway. Ku, a heterodimer consisting of Ku70 and Ku80, functions in the recognition step of this pathway through binding DNA termini. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to create the full DNA-PK heterotrimer. Formation of DNA-PK results in autophosphorylation as well as phosphorylation of downstream proteins of the NHEJ pathway. Previous work shows that the extreme C-terminus of Ku80 stimulates the kinase activity of DNA-PKcs, and Ku DNA binding is regulated as a function of redox via stimulation of a conformational change when oxidized resulting in a decrease in DNA binding activity. To further understand these methods of regulation of Ku and DNA-PK, a pair of mutants has been constructed; one consisting of full length Ku70 and truncated Ku80 (Ku70/80ΔC) lacking 182 C-terminal amino acids. The removal of these amino acids was shown to have little to no effect on the proteins expression, stability or DNA binding, as determined by SDS-PAGE, western blot analysis and electrophoretic mobility shift assay (EMSA). When oxidized Ku70/80ΔC showed a decrease in DNA binding similar to that seen in wild type, however when re-reduced the mutant did not recover to the same extent as wild type. A second mutant was constructed, containing amino acids 590-732 of Ku80 (Ku80CTR), to further understand the mechanism by which Ku80 C-terminus interacts with the rest of the Ku heterodimer. Possible protein-protein interactions were evaluated by Ni-NTA affinity, gel filtration chromatography, fluorescence polarization and two forms of protein-protein cross-linking. Ni-NTA agarose affinity, and gel filtration chromatography failed to reveal an interaction in the presence or absence of DNA. However, photo-induced cross-linking of unmodified proteins (PICUP) as well as EDC cross-linking demonstrated an interaction which was not affected by DNA. The work presented here demonstrates that the interaction between Ku80CTR and Ku is rather weak, but it does exist and plays a relatively large role in the NHEJ pathway.