In order to preserve its genomic integrity, an organism needs to detect and repair DNA double-strand breaks (DSBs) in a prompt and accurate fashion. This goal is accomplished by enabling an exquisitely sensitive DSB sensing apparatus as well as multiple and often overlapping pathways for repair. All of these processes are carried out on a highly organized and compacted chromatin substrate in the nucleus. An important question is whether chromatin plays an active role in the process and whether it helps in the signaling or repair of this damage. We have used Chromosome Conformation Capture (3C) to show that there are no large scale changes in chromosome structure at a single site-specific DNA double-strand break, although looping interactions between DSBs and donors can be detected. In a surprising result, we found that 3C detected a nucleus-wide decrease in interactions with the DSB. We have used a combination of 3C, fluorescence microscopy and chromatin immunoprecipitation to show that the decrease in interactions is a result of the relocalization of persistent DSB to the nuclear periphery. We also show that this is dependent on the recruitment of telomerase complex to the DSB, which then interacts with its natural partner in the Inner nuclear membrane, Mps3, and relocalizes the DSB to the periphery. Thus, a DSB that cannot be repaired is shunted into a pathway where the cell attempts to survive by putting a de novotelomere on the broken chromosome. Remarkably, this is not an irreversible phenomenon despite the recruitment of telomerase and the relocalization to the periphery. DSBs which are repaired slowly due to the presence of homology on a different chromosome, or merely usage of a kinetically slower form of repair, undergo this pathway switch, but can still recover and repair the DSB if homology is present. We also show that the role of the periphery is to ensure repair through de novotelomere formation or other non-canonical repair pathways. Indeed, loss of peripheral localization results in a dramatic suppression of the genomic instability of the Slx5/8 mutants, which have been implicated in the persistent DSB response at the Nuclear pores. Thus, the nuclear periphery is a special compartment where DSBs go after they cannot be repaired by canonical pathways. Specialized components such as telomerase, silencing proteins and components of the SUMO pathway, all seem to play roles in the healing of these chromosomes. Importantly, the SUN domain homologues of Mps3 have been shown to play roles similar to their yeast homologues in meiotic bouquet formation through their interactions with telomeres. Thus, they may represent a conserved mechanism for chromosome healing and telomere anchoring, despite the fact that mammalian telomeres are rarely found at the nuclear periphery. Such survival mechanisms may be expected to operate in cancer cells which may or may not have upregulated telomerase expression.

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Résumé : Chez toutes les cellules, une modulation précise de l’expression des gènes est essentielle afin de réguler adéquatement leur métabolisme et de s’adapter aux changements environnementaux. En effet, c’est l’expression des gènes, plutôt que la séquence d’ADN, qui détermine en grande partie la diversité et la complexité des organismes. Celle-ci dépend principalement des changements dans les niveaux d’ARNs cellulaires résultant de la modification de l’équilibre entre leurs taux relatifs de synthèse et de dégradation. Alors que la régulation transcriptionnelle a été largement étudiée par le passé, des études récentes révèlent que la stabilité de l’ARN joue aussi un rôle important dans le modelage du transcriptome. Toutefois, les mécanismes qui assurent la dégradation précise et sélective des ARNs sont globalement mal compris. Au cours de cette thèse, j’ai utilisé la ribonucléase III de levure Saccharomyces cerevisiae (Rnt1p) comme modèle pour étudier comment des transcrits spécifiques sont ciblés pour la dégradation et évaluer sa contribution à la régulation de l’expression génique. Les résultats indiquent que Rnt1p régule l’expression des gènes en utilisant une spécificité élargie pour des structures tige-boucles d’ARN. En effet, un nouveau motif structurel de Rnt1p permet la discrimination des tige-boucles ayant une séquence spécifique tout en bloquant la liaison à des hélices génériques d’ARN double-brin. D’un autre côté, l’identification des signaux de dégradation de Rnt1p à l’échelle du transcriptome a permis de révéler plus de 384 transcrits clivés par Rnt1p, dont la majorité sont des ARN messagers. En outre, l’impact de la délétion de RNT1 sur l’expression de ces gènes est influencé par les conditions de culture des cellules, ce qui suggère que Rnt1p est un important régulateur conditionnel de l’expression génique. Somme toute, les résultats présentés dans cette thèse démontrent comment des ARNs sont spécifiquement choisis pour la dégradation et soulignent l’importance de la dégradation nucléaire dans la régulation de l’expression génique en réponse à des changements environnementaux. // Abstract : Precise modulation of gene expression is essential for any cell in order to regulate its metabolism and adapt to environmental changes. In fact, it is gene expression, rather than DNA sequence alone, which mostly explains the functional diversity and complexity between the different cell types. As such, gene expression mainly results from changes in the levels of cellular RNAs which are, in turn, dependent on the equilibrium between their relative rates of synthesis and degradation. While transcriptional control has been largely studied in the past, recent publications reveal that changes in RNA stability also play an important role in shaping the transcriptome. Unfortunately though, the mechanisms ensuring precise and selective RNA degradation remains poorly understood. In this thesis, I have used the yeast Saccharomyces cerevisiae ribonuclease III (Rnt1p) as a model to study how specific transcripts are targeted for degradation and evaluate its contribution to the regulation of gene expression. The results indicate that Rnt1p regulates gene expression using a broad specificity for structured RNA stem loops. Indeed, a new structural motif of Rnt1p permits discrimination of hairpins with specific sequence while blocking the binding of the generic RNA duplexes recognized by other members of the RNase III family. This highly specific mode of substrate recognition was found to be easily modulated by a flexible network of protein RNA interactions. On the other hand, transcriptome-wide identification of Rnt1p degradation signals uncovered more than 384 transcripts, including 291 mRNAs. Interestingly, the impact of RNT1 deletion on mRNA expression is modulated by changes in the growth conditions of the cell, indicating that Rnt1p is an important regulator of conditional gene expression. Overall, the results presented in this thesis demonstrate how specific RNAs are selected for degradation and highlight the importance of nuclear RNA decay for fine tuning gene expression in response to changes in growth conditions.

Activation-induced cytidine deaminase (AID) is a key protein required for both class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes. AID is induced in B cells during an immune response. Lack of AID or mutant form of AID causes immunodeficiency; e.g., various mutations in the C terminus of AID causes hyper IgM (HIGM2) syndrome in humans. The C terminal 10 amino acids of AID are required for CSR but not for SHM. During both CSR and SHM, AID deaminates dCs within Ig genes, converting them to dUs, which are then either replicated over, creating mutations, or excised by uracil DNA glycosylase (UNG), leading to DNA breaks in Ig switch regions. Also, the mismatch repair (MMR) heterodimer Msh2-Msh6 recognizes U:G mismatches resulting from AID activity and initiates MMR, which leads to increased switch region double strand breaks (DSBs). DSBs are essential intermediates of CSR; lack of UNG or MMR results in a reduction of DSBs and CSR. The DSBs created in the Sμ and one of the downstream S-regions during CSR are recombined by non-homologous end joining (NHEJ) to complete CSR. Available data suggest that AID is required not only for the deamination step of CSR, but also for one or more of the steps of CSR that are downstream of deamination step. This study investigates the role of C terminus of AID in CSR steps downstream of deamination. Using retroviral transduction into mouse splenic B cells, I show that AID binds cooperatively with UNG and Msh2-Msh6 to the Ig Sμ region, and this depends on the AID C terminus. I also show that the function of MMR during CSR depends on the AID C terminus. Surprisingly, the C terminus of AID is not required for Sμ or Sγ3 DSBs, suggesting its role in CSR occurs during repair and/or recombination of DSBs.

Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.

Chez les plantes, le génome plastidique est continuellement exposé à divers stress mutagènes, tels l’oxydation des bases et le blocage des fourches de réplication. Étonnamment, malgré ces menaces, le génome du plastide est reconnu pour être très stable, sa stabilité dépassant même celle du génome nucléaire. Néanmoins, les mécanismes de réparation de l’ADN et du maintien de la stabilité du génome plastidique sont encore peu connus. Afin de mieux comprendre ces processus, nous avons développé une approche, basée sur l’emploi de la ciprofloxacine, qui nous permet d’induire des bris d’ADN double-brins (DSBs) spécifiquement dans le génome des organelles. En criblant, à l’aide de ce composé, une collection de mutants d’Arabidopsis thaliana déficients pour des protéines du nucléoïde du plastide, nous avons identifié 16 gènes vraisemblablement impliqués dans le maintien de la stabilité génomique de cette organelle. Parmi ces gènes, ceux de la famille Whirly jouent un rôle primordial dans la protection du génome plastidique face aux réarrangements dépendants de séquences de microhomologie. Deux autres familles de gènes codant pour des protéines plastidiques, soit celle des polymérases de types-I et celle des recombinases, semblent davantage impliquées dans les mécanismes conservateurs de réparation des DSBs. Les relations épistatiques entre ces gènes et ceux des Whirly ont permis de définir les bases moléculaires des mécanismes de la réparation dépendante de microhomologies (MHMR) dans le plastide. Nous proposons également que ce type de mécanismes servirait en quelque sorte de roue de secours pour les mécanismes conservateurs de réparation. Finalement, un criblage non-biaisé, utilisant une collection de plus de 50,000 lignées mutantes d’Arabidopsis, a été réalisé. Ce criblage a permis d’établir un lien entre la stabilité génomique et le métabolisme des espèces réactives oxygénées (ROS). En effet, la plupart des gènes identifiés lors de ce criblage sont impliqués dans la photosynthèse et la détoxification des ROS. Globalement, notre étude a permis d’élargir notre compréhension des mécanismes du maintien de la stabilité génomique dans le plastide et de mieux comprendre l’importance de ces processus. / The plant plastidial genome is constantly threatened by many mutagenic stresses, such as base oxidation and replication fork stalling. Despite these threats, the plastid genome has long been known to be more stable than the nuclear genome, suggesting that alterations of its structure would have dramatic consequences on plant fitness. At the moment, little is known about the genes and the pathways allowing such conservation of the organelle genome sequences. To gain insight into these mechanisms, we developed an assay which uses ciprofloxacin, a gyrase inhibitor, to generate DNA double-strand breaks (DSBs) exclusively in plant organelles. By screening mutants deficient for proteins composing the plastid nucleoid on ciprofloxacin, we were able to identify 16 candidate genes, most likely involved in the repair of DSBs in plastid. Among these genes, those of the Whirly family of single-stranded DNA binding proteins are shown to be key factors in protecting the genome from error-prone microhomology mediated repair (MHMR). Two other family of proteins, the plastid type-I polymerases and the plastid recombinases, seem to be involved in the conservative repair pathways. The evaluation of the epistatic relationship between those two genes and the Whirly genes led us to define the molecular basis of MHMR and to propose that they might act as a backup system for conservative repair pathways. Finally, a non-biased screen, using 50,000 different insertion lines, allowed the identification of numerous genes that were already associated with ROS homeostasis, suggesting a link between DNA repair and ROS imbalance. Globally, our study shed light on the mechanisms that allow the maintenance of plastid genome, while explaining the importance of such conservation of the plastid genome.

In eukaryotes, the repair of DSBs is accomplished through two broadly defined processes: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The central step of HR is pairing and exchange of strands between two homologous DNA molecules, which is catalyzed by the conserved Rad51/RecA family of proteins. Prior to this step, an essential step in all HR pathways i.e. 5'→3' resection of broken DNA ends to generate 3' single stranded DNA tails. At the molecular level, initiation of DNA end resection is accomplished through the concerted action of MRX complex (Mre11, Rad50 and Xrs2) and Sae2 protein. To elucidate the molecular basis underlying DSB end resection in S. cerevisiae mre11 nuclease deficient mutants, we have performed a comprehensive analysis of the role of S. cerevisiae Mre11 (henceforth called as ScMre11) in the processing of DSB ends using a variety of DNA substrates. We observed that S. cerevisiae Mre11(ScMre11) exhibits higher binding affinity for single- over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Furthermore, reconstitution of DSB end resection network in-vitro revealed that Rad50, Xrs2, and Sae2 potentiated the DNA unwinding activity of Mre11. Since the exonuclease activity of Mre11 is of the opposite polarity to that expected for resection of DSBs, unwinding activity of Mre11 in conjunction with Rad50, Xrs2, and Sae2 might provide an alternate mechanism for the generation of ssDNA intermediates for DSB end repair and HR. Additionally, ScMre11 displays strong homotypic as well as heterotypic interaction with Sae2. In summary, our results revealed important insights into the mechanism of DSB end processing and support a model in which Sae2, Rad50, and Xrs2 positively regulate the ScMre11-mediated DNA unwinding activity via their direct interactions or through allosteric effects on the DNA or cofactors. Prompted by the closer association of MRX and Sae2 during DSB end processing, we asked whether Sae2 and its endonuclease activity is required for cellular response to replication stress caused by DNA damage. Toward this end, we examined the sensitivity of S. cerevisiae wild type, sae2Δ and various SAE2 mutant strains defective in phosphorylation and nuclease activity in the presence of different genotoxic agents, which directly or indirectly generate DSBs during replication. We found that S. cerevisiae lacking SAE2 show decreased cell viability, altered cell cycle dynamics after DNA damage, and more specifically, that Sae2 endonuclease activity is essential for these biological functions. To corroborate the genetic evidences for role of SAE2 during replicative stress, we investigated SAE2 functions in-vitro. For this, we purified native Sae2 protein and nuclease dead mutant of Sae2 i.e. sae2G270D. Our studies revealed dimeric forms of both the wild type and mutant forms of Sae2. Furthermore, Sae2 displays higher binding affinity and catalytic activity with branched DNA structures, such as Holliday junction and replication forks. By using nuclease dead Sae2 protein i.e. sae2G270D, we confirmed that the endonuclease activity is not fortuitous and is intrinsic to Sae2 polypeptide. Furthermore, nuclease-defective Mre11 stimulates Sae2endonuclease activity. Mapping of the cleavage sites of Sae2 revealed a distinct preference for cleavage on the 5' end of the Holliday junction, suggesting the importance of Sae2 nuclease during recombination mediated restart of the reversed replication fork. In summary, our data clearly demonstrate a previously uncharacterized role for Sae2 nuclease activity in resection of DSB ends, processing of intermediates of DNA replication/repair and attenuation of DNA replication stress-related defects in S. cerevisiae.

Elastic properties of carbon nanotubes (CNT), boron nitride nanotubes (BNNT), double stranded DNA (dsDNA), paranemic-juxtapose crossover (PX-JX) DNA and dendrimer bound DNA are discussed in this thesis. Structural phase transitions of nucleic acids induced by external force, carbon nanotubes and graphene substrate are also studied extensively. Electrostatic interactions have a strong effect on the elastic properties of BNNTs due to large partial atomic charges on boron and nitrogen atoms. We have computed Young’s modulus (Y ) and shear modulus (G) of BNNT and CNT as a function of the nanotube radius and partial atomic charges on boron and nitrogen atoms using molecular mechanics calculation. Our calculation shows that Young’s modulus of BNNTs increases with increase in magnitude of the partial atomic charges on B and N atoms and can be larger than the Young’s modulus of CNTs of same radius. Shear modulus, on the other hand depends weakly on the magnitude of partial atomic charges and is always less than the shear modulus of the CNT. The values obtained for Young’s modulus and shear modulus are in excellent agreement with the available experimental results. We also study the elasticity of dsDNA using equilibrium fluctuation methods as well as nonequilibrium stretching simulations. The results obtained from both methods quantitatively agree with each other. The end-to-end length distribution P(ρ) and angle distribution P(θ) of the dsDNA has a Gaussian form which gives stretch modulus (γ1) to be 708 pN and persistence length (Lp) to be 42 nm, respectively. When dsDNA is stretched along its helix axis, it undergoes a large conformational change and elongates about 1.7 times its initial contour length at a critical force. Applying a force perpendicular to the DNA helix axis, dsDNA gets unzipped and separated into two single-stranded DNA (ssDNA). DNA unzipping is a fundamental process in DNA replication. As the force at one end of the DNA is increased the DNA starts melting above a critical force depending on the pulling direction. The critical force fm , at which dsDNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the dsDNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base-pairs. Similar force-extension curve has also been observed when crossover DNA molecules are stretched along the helix axis. In the presence of mono-valent Na+ counterions, we find that the stretch modulus (γ1 ) of the paranemic crossover (PX) and its topoisomer juxtapose (JX) DNA structure is significantly higher (30 %) compared to normal B-DNA of the same sequence and length. When the DNA motif is surrounded by a solvent of divalent Mg2+ counterions, we find an enhanced rigidity compared to in Na+ environment due to the electrostatic screening effects arising from the divalent nature of Mg2+ counterions. This is the first direct determination of the mechanical strength of these crossover motifs which can be useful for the design of suitable DNA motifs for DNA based nanostructures and nanomechanical devices with improved structural rigidity. Negatively charged DNA can be compacted by positively charged dendrimer and the degree of compaction is a delicate balance between the strength of the electrostatic interaction and the elasticity of DNA. When the dsDNA is compacted by dendrimer, the stretch modulus, γ1 and persistence length, Lp decreases dramatically due to backbone charge neutralization of dsDNA by dendrimer. We also study the effect of CNT and graphene substrate on the elastic as well as adsorption properties of small interfering RNA (siRNA) and dsDNA. Our results show that siRNA strongly binds to CNT and graphene surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the CNTs and is maximum on graphene. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the CNT/graphene surface. However, dsDNA of the same sequence undergoes much less unzipping and wrapping on the CNT/graphene due to smaller interaction energy of thymidine of dsDNA with the CNT/graphene compared to that of uridine of siRNA. Unzipping probability distributions fitted to single exponential function give unzipping time (τ) of the order of few nanoseconds which decrease exponentially with temperature. From the temperature variation of unzipping time we estimate the free energy barrier to unzipping. We have also investigated the binding of siRNA to CNT by translocating siRNA inside CNT and find that siRNA spontaneously translocates inside CNT of various diameters and chiralities. Free en- ergy profiles show that siRNA gains free energy while translocating inside CNT and the barrier for siRNA exit from CNT ranges from 40 to 110 kcal/mol depending on CNT chirality and salt concentration. The translocation time τ decreases with the increase of CNT diameter having a critical diameter of 24 A for the translocation. After the optimal binding of siRNA to CNT/graphene, the complex is very stable which can serve as siRNA delivery agent for biomedical applications. Since siRNA has to undergo unwinding process in the presence of RNA-induced silencing complex, our proposed delivery mechanism by single wall CNT possesses potential advantages in achieving RNA interference (RNAi).

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