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Résumé : Les télomères sont des structures nucléoprotéiques spécialisées qui assurent la stabilité du génome en protégeant les extrémités chromosomiques. Afin d’empêcher des activités indésirables, la réparation des dommages à l’ADN doit être convenablement régulée au niveau des télomères. Pourtant, il existe peu d’études de la réparation des dommages induits par les ultraviolets (UVs) dans un contexte télomérique. Le mécanisme de réparation par excision de nucléotides (NER pour « Nucleotide Excision Repair ») permet d’éliminer les photoproduits. La NER est un mécanisme très bien conservé de la levure à l’humain. Elle est divisée en deux sous voies : une réparation globale du génome (GG-NER) et une réparation couplée à la transcription (TC-NER) plus rapide et plus efficace. Dans notre modèle d’étude, la levure Saccharomyces cerevisiae, une forme compactée de la chromatine nommée plus fréquemment « hétérochromatine » a été décrite. Cette structure particulière est présente entre autres, au niveau des régions sous-télomériques des extrémités chromosomiques. La formation de cette chromatine particulière implique quatre protéines nommées Sir (« Silent Information Regulator »). Elle présente différentes marques épigénétiques dont l’effet est de réprimer la transcription. L’accès aux dommages par la machinerie de réparation est-il limité par cette chromatine compacte ? Nous avons donc étudié la réparation des lésions induites par les UVs dans différentes régions associées aux télomères, en absence ou en présence de protéines Sir. Nos données ont démontré une modulation de la NER par la chromatine, dépendante des nucléosomes stabilisés par les Sir, dans les régions sous-télomériques. La NER était moins efficace dans les extrémités chromosomiques que dans les régions plus proches du centromère. Cet effet était dépendant du complexe YKu de la coiffe télomérique, mais pas dépendant des protéines Sir. La transcription télomériques pourrait aider la réparation des photoproduits, par l’intermédiaire de la sous-voie de TC-NER, prévenant ainsi la formation de mutations dans les extrémités chromosomiques. Des ARN non codants nommés TERRA sont produits mais leur rôle n’est pas encore clair. Par nos analyses, nous avons confirmé que la transcription des TERRA faciliterait la NER dans les différentes régions sous-télomériques. / Abstract : Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. DNA lesions constitute a serious risk to genome integrity. Thus, DNA repair mechanisms are required for continuous and unabridged cell divisions. The nucleotide excision repair (NER) pathway removes bulky DNA lesions such as UV-induced photoproducts, like the cyclobutane pyrimidine dimers (CPD). NER is divided in two sub-pathways: global genome repair (GGR) and the faster transcription-coupled repair (TCR), which only differ in how they recognize UV-induced lesions. In eukaryotes, NER must find and repair DNA lesions that are buried in nucleosomes. In the yeast S. cerevisiae, genes positioned close to telomeres are silenced by a heterochromatin-like structure that is formed by silent information regulator proteins (Sir). To determine if nucleosomes and chromatin in subtelomeric regions affect the efficiency of NER, we studied the repair of photoproducts in different telomere-associated regions in both, WT and SIR genes deleted cells (sirΔ). We found that NER efficiency was modulated by the presence of nucleosomes on the subtelomeric type X element. In addition, in absence of Sir proteins, NER efficiency increased and was not modulated by nucleosomes, indicating that nucleosome positioning was less defined in sirΔ cells. Remarkably, in telomeric restriction fragment, NER was less efficient at telomeres than in the subtelomere type Y’ element. We suggest that low NER efficiency at the very end of chromosomes results from attachment sites to the nuclear periphery. Our data indicate that NER in sub-telomeric chromatin is modulated by Sir proteins stabilized-nucleosomes, and that NER is inhibited in telomeric chromatin by the presence of YKu, independently from the presence of Sir proteins. It was recently shown that the chromosome ends are transcribed and a non-coding RNA, called TERRA, is produced. Currently the precise functions of TERRA are not understood. Our second goal is to help understand the function of TERRA. We think that transcription at the chromosome ends could facilitate the removal of DNA lesions from heterochromatin by TCR, which would prevent the formation of mutations and, ultimately, chromosome shortening. Our data showed that TC-NER is effective in Y’ element and the telomere. Without Sir proteins, TERRA transcription is found in a particular region at the end of the X element. The transcription of TERRA could improve the repair of UV-induced lesions.

Background and Aims: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation–induced T cell dysfunctions during HCV infection remain elusive. Approach and Results: Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+. In contrast, the expression of stem cell–like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. Conclusions: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.

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Chronic hepatitis C virus (HCV) infection exhibits persistent high viral load, inducing T cells differentiation and dysfunction in chronically infected individuals. Recent longitude studies in both HCV specific- and bulk T cells reveal that chronic immune stimulation is the driving force for the impaired T cell functions, however, the underlying mechanisms remain elusive. Here, we show that peripheral CD4+ T cells from chronically HCV-infected patients exhibit lymphopenia with the reduction of naïve population and expansion of effector memory T cells. CD4+ T cells from HCV patient also display elevated activation markers. including HLA-DR, GLUT1, Granzyme B, and short-lived effector marker CD127- KLRG1+, whereas stem cell-liked transcription factor TCF1 and telomere sheterin subunit TRF2 are significant reduced, comparing to age- and gender-matched healthy controls. Mechanistically, ex vivo T cell differentiation revealed that CD4+ T cells from HCV patients exhibit PI3K/Akt/mTOR signaling hyperactivation upon TCR stimulation, favoring pro-inflammatory effector differentiation with TRF2 downregulation, rendering telomere dysfunction induced foci (TIFs) accumulation, resulting in telomeric DNA damage and cellular apoptosis. Importantly, exacerbation of telomere deprotection by knockdown of TRF2 expression in healthy T cells resulted in an increase in telomeric DNA damage and T cell apoptosis; whereas overexpression of TRF2 in HCV-T cells led to an alleviation of telomeric DNA damage and T cell death. Additionally, inhibition of Akt signaling during T cell activation can preserve precursor memory population, while limiting inflammatory effector expansion, DNA damage, and cell death. Taken together, these results suggest that modulation of immune activation by inhibiting Akt signaling and protection of telomeres by enforcing TRF2 expression could open new therapeutic strategies to balance adaptive immune responses in the setting of chronic immune activation and inflammatory in vulnerable populations such as chronically viral infected individuals.

The study for surfaced-immobilized nucleic acid probes in nanometer region in response to hybridization and to discrimination ofdifferent target nuclei acids. The hairpin locked nucleic acid (LNA-HP) isselected to be the probe molecule, and target molecules include perfect complementary (PC) and single mismatch (1MM). The self-assembledLNA-HP molecular nanospot is successfully prepared by liquid phaseAFM (Atomic Force Microscope)-based nanolithography technique, then in situ hybridization is carried out by using different targets (PC/1MM).To obtain the information of structure change, we use AFM to analyze therelative heights in the process of hybridization. The experimental results point out that (1) the structure changes of surface probe molecules maycorrelate with the AFM signal when target sequence hybridizes to the probe, (2) miniaturization of the size of the nucleic acid probe may promote hybridization efficiency and enhance the discrimination between PC and 1MM. Studies on whether the different chemical impetus in solution can affect conformation of the human telomeric DNA of sequence is conducted. A human talomeric DNA composed of ( 5¡¦-TTAGGG-3¡¦:5¡¦-CCCTAA-3¡¦ ) repeats, with a 100-200 nt ( T2AG3 ) repetitive unit overhang at 3¡¦ ends is chosen. This extended single-stranded sequence is called G-rich DNA, which forms the special G-quadruplex structure in solution containing sodium ions or potassium ions. The single-stranded sequence composed of ( C3TA2 ) repetitive units called C-rich DNA displays the i-motif folded structure in the low pH environment. These biomimetic DNA¡¦s are thiol-modified to self-assemble on gold surfaces. Separate measurements with AFM (the molecular thickness and rootmean- square roughness of the self-assembly monolayer of DNA ) and CD( circular dichroism ) ( structure characterization ) confirm the conformational changes of G-rich and C-rich DNA¡¦s on gold surface are indeed dependent of the presence of cations and protons.

Meiotic recombination is a prerequisite for exchange of genetic information in all Sexually reproducing organisms. This process is initiated by the formation of double stranded breaks (DSBs) in DNA followed by homology directed repair. The process is subjected to surveillance mechanisms that control DSB formation and allow for repair of DSBs by halting cell cycle progression. Interestingly, though generation of DSBs is an Essential event in meiosis they are nevertheless regarded as the most lethal forms of DNA damage. If left unrepaired a single DSB can lead to gene deletion, duplication, translocations and missegregation of large chromosome fragments leading to cell death. In Saccharomyces cerevisiae, genetic screens for mutants defective in meiotic recombination led to the identification of a group of genes called the RAD52 epistasis group which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2. A subset of these genes, namely MRE11, RAD50 and XRS2, have been shown by genetic studies to be essential for several nuclear events including sensing DSBs, double strand break repair (DSBR) by homologous recombination (HR) and non homologous end joining (NHEJ), telomere length maintenance, cell cycle activation in response to DSBs, mitotic and meiotic recombination. In vitro, Mre11 displays Mn2+-dependent endonuclease activity on ssDNA, 3'-5' Exonuclease on single- and double-stranded DNA, strand annealing and weak hairpin Opening activities. Mutational analyses have revealed two functional domains in Mre11- Then terminal nuclease domain involved in telomere length maintenance and DSB Processing and the C terminal DNA binding domain involved in DSB formation during Meiosis. Rad50, a 153 kDa protein shares homology with the SMC (Structural Maintenance of Chromosome) family of proteins which are involved in chromosome Condensation and cohesion. It consists of a bipartite N- and C terminal Walker A and Walker B motifs separated by a heptad repeat sequence which folds into an antiparallel Coiled-coil structure. The heptad repeats are separated by a metal binding globular region the Zn hook. Rad50 is an ATP-dependent DNA-binding protein. hRad50 regulates the exonuclease activity of hMre11. Unlike Mre11 and Rad50, which are evolutionarily conserved, Xrs2 is found only in S. cerevisiae and Nbs1 in mammals. Xrs2 appears to be sequence non-specific DNA- binding protein. Xrs2 in yeast or Nbs1 is its counterpart in mammals target Mre11 and Rad50 to the sites of DNA damage and mediate S-phase cell cycle checkpoint activation. Mutations in either one of the MRX subunits results in defects in repair of DSBs, activation of cell cycle checkpoint and shortened telomeres leading to genomic instability. Hypomorphic mutations in MRE11 and NBS1 lead to genetic disorders- A-TLD (ataxia-telangiectasia-like disorder) and NBS (Nijmegen breakage syndrome) respectively, that are phenotypic ally related to AT (ataxia-telangiectasia) caused by mutations in ATM. Patients with AT, A-TLD or NBS syndromes are hypersensitive to radiomimetic agents and are predisposed to cancer. Several lines of evidence suggest that S. cerevisiae strains bearing mre11Δ, rad50Δ or xrs2Δ display shortening of telomeres. Telomeres are the nucleoprotein ends of all linear eukaryotic chromosomes that are important in maintaining the integrity of the genome.Telomeres are comprised of repetitive G rich sequence most of which is double stranded but the extreme 3' end protrudes to form 3' single stranded overhang called the G tail. elopers are essential in preventing end-end fusion of chromosome, are important for chromosome replication, segregation and genome stability. Genetic studies have implicated the MRX complex in both telomerase-dependent and independent telomere length maintenance. Studies have indicated a direct role for S. cerevisiae MRE11 in the proper establishment of telomere end-structure. However, the molecular mechanism of MRX at telomeres is poorly understood. To understand the role(s) of MRX complex at telomeres, it is important to elucidate the biochemical activities of MRX complex as well as its individual subunits on the telomere DNA structures. Since, Mre11 complex is known to function in several processes related to DNA metabolism it becomes imperative to study the function of Mre11 complex on DNA substrates in the context of a given nuclear process. The 3' single trended telomeric sequence is capable of acquiring folded conformation(s) as a mechanism of end protection which is mediated by several telomere-specific and nonspecific ending proteins. In mammals, the 3' ssDNA has been demonstrated to fold into tloop configuration mediated by some of the components of sheltrin protein complex, wherein the ssDNA invades the duplex DNA resulting in the formation of a displacement loop (D loop). Evidence for the formation of t-loop has been shown in vitro with human telomeres. However, the formation of t-loops has not been demonstrated in S. cerevisiae. Nevertheless, there is growing body of evidence which suggests the formation of alternative DNA structures such as G4 DNA at the yeast telomeres. G quadruplexes (G quartets or G4 DNA) are thermodynamically stable structures formed by Hoogsteen base pairing between guanine residues. In a G quartet the four guanine residues are paired, where each guanine residue is an electron acceptor and a donor and stabilized by a metal cation. The presence of G rich motifs at the promoter regions, rDNA, telomeres and recombination hot spots indicate that G4 DNA has important functions in vivo. Although the existence of G4 DNA has been the subject of much debate, the identification of several proteins that promote (Rap1, Hop1, Topo I, TEBPβ), modify and resolve (POT1, TERT, KEM1, GQN1, BLM, WRN, Rte1) G4 DNA, together with the direct visualization of G4 DNA using G4 DNA specific antibodies and RNA interference have provided compelling for the existence of G4 DNA in vivo. To elucidate the function of MRX complex or its individual subunits at telomeres, the biochemical activities of purified MRX complex and its individual subunits on G4 DNA, D loop, duplex DNA and G rich ssDNA has been analyzed in this study. G4 DNA was assembled from S. cerevisiae telomeric sequence. G4 DNA was isolated and its identity was ascertained by chemical probing and circular dichroism. S. cerevisiae MRE11 and XRS2 was cloned and expressed in E. coli BL21 (DE3)plysS. S. cerevisiae RAD50 in pPM231 vector in S. cerevisiae BJ5464 strain was a gift from Dr. Patrick Sung (Yale University). Mre11, Rad50 and Xrs2 were overexpressed and purified to >98% homogeneity. The identity of the proteins was ascertained by Western bloting using polyclonal antibodies. Using purified proteins heterotrimeric MRX and heterodimeric MR and MX protein complexes were formed in the absence of ATP, DNA or Mn2+. The ability of M/R/X to bind to telomeric DNA substrates was studied by electrophoretic mobility shift assays. Mre11, Rad50, Xrs2 and MRX displayed higher binding affinity for G4 DNA over D loop, ss- or dsDNA. MRX bound G4 DNA more efficiently compared to its individual subunits as 10-fold lower concentration of MRX was able to shift the DNA into the protein-DNA complex. The protein-G4 DNA complexes were stable as >0.8 M NaCl as required to dissociate 50% of protein-G4 DNA complexes. Efficient competition by poly(dG), which is known to fold into G4 DNA, suggested that the protein-G4 DNA complex was specific. Competition experiments with tetra-[N-methyl- pyridyl]-porphyrin suggested that M/R/X recognizes distinct determinants and makes specific interactions with G4 DNA. G4 DNA is highly polymorphic and can exist as intramolecular or intermolecular (parallel and antiparallel) structures. High affinity binding of Mre11 to G4 DNA (parallel) over G2' DNA (antiparallel), ss- and dsDNA suggests the existence of parallel G4 DNA structures at the telomeres and that G4 DNA may be the natural substrate for MRX complex in vivo. Telomeres are elongated by telomerase that requires access to the 3' G-tail for its activity. Formation of G4 DNA structures renders the 3' G-tail inaccessible to telomerase thereby inhibiting telomere elongation. To elucidate the functional relevance of high affinity of M/R/X for G4 DNA, the ability of the complex to generate the appropriate DNA structure for telomere elongation has been analyzed. In this study, I considered the possibility that MRX could act as: (a) a helicase that opens up the G4 DNA structures making it accessible to telomerase or (b) as a nuclease that cleaves the G4 DNA generating substrates for telomerase. Helicase assay with Mre11, Xrs2, MX and MRX on G4 DNA and duplex DNA showed no detectable DNA unwinding activity. Interestingly, nuclease assays with Mre11 on G4 DNA showed that Mre11 cleaved G4 DNA in Mn2+-dependent manner and the cleavage was mapped to the G residues at the stacks of G quartets. Mre11 cleaved telomeric duplex DNA in the center of TGTG repeat sequence, G rich ssDNA at 5' G residue in an array of 3 G residues and D loop structure preferentially at the 5' ends at TG residues. Significantly, the endonuclease activity of Mre11 was abrogated by Rad50. Xrs2 had no effect on the endonuclease activity of Mre11. Structural studies on Rad50 and Mre11 showed that binding of ATP by Rad50 positions the Rad50 catalytic domain in close proximity to the nuclease active site of Mre11. In yeast, disruption of ATP binding Walker motifs results in a null phenotype, suggesting that ATP is required for Rad50 functions in vivo. hRad50 is known to regulate the exonuclease activity of hMre11 in the presence of ATP. Therefore, can ATP modulate the effect of S. cerevisiae (Sc) Rad50 on ScMre11? To address this question, I monitored the ATPase activity of Rad50 in the absence or presence of DNA. Rad50 hydrolyzed ATP in a DNA-independent manner; however, ATPase activity was enhanced in the presence of Mre11 and Xrs2. However, Rad50 exhibited a low turnover indicating that ATP could function as a switch molecule. Based on these observations, the effect of ATP on the nuclease activity was examined. The binding of ATP and its hydrolysis by Rad50 attenuated the inhibition exerted by Rad50 on the Mre11 endonuclease activity. Cleavage of G4 DNA, D loop, duplex DNA and ssDNA required ATP hydrolysis, since no cleavage product was observed when ADP or ATPγS was substituted for ATP. This observation was corroborated using a hairpin DNA substrate that mimics a intermediate in VDJ recombination, thereby confirming the generality of regulation of Rad50 on the endonuclease activity of Mre11. Does Rad50 regulate the exonuclease activity of Mre11 as well? To address this question, exonuclease activity of Mre11, MR and MRX on 3' labeled duplex DNA and G4 DNA was assayed. Rad50 had no measurable effect on the exonuclease activity of Mre11. Based on previous studies and my observations, I propose a model for the role of MRX in telomere length maintenance and its regulation by the ATP-binding pocket of Rad50. MRX binds telomeric DNA substrates in a non-productive complex, which is converted to a catalytically active complex upon binding of ATP by Rad50. ATP induces conformational changes, repositioning the complex such that the catalytic site of Mre11 now has access to the substrate. Following cleavage of DNA by Mre11, the release of ADP and inorganic phosphate, generate the cleaved product. The cleaved DNA is now accessible to telomerase or telomere binding proteins. In summary, the data presented in my PhD thesis demonstrates that Mre11 is a structure- and sequence-specific endonuclease. The natural substrate for telomerase is the 3' ssDNA. G quartets at telomeres not only protect the ends from degradation but also make the ends inaccessible for telomerase activity. Genetic studies have shown that cells proficient for telomerase activity but lacking any one of the components of the MRX complex display shortening in telomere length. The ability of Mre11 to cleave G4 DNA at the stacks of G quartets therefore, suggests a mechanism by which the 3' ssDNA is rendered accessible to telomerase or other telomere binding proteins. Yeast telomeres are characterized by the presence of subtelomeric Y' elements proximal to the terminal TG1- 3 repeat sequences. The Y' element has been shown to be amplified by telomerase in a fraction of mutants with short telomeres. The mechanism by which Y' DNA is amplified is unclear. The ability of Mre11 to cleave telomere duplex DNA at the center of TGTG repeats could contribute to the generation of appropriate substrate for elongation by telomerase, thereby contributing to Y' DNA amplification. Telomere length is maintained by homeostasis between processes that contribute to telomere elongation and those that cause attrition in telomeric ends. Overelongated telomeres are brought to wild type telomere size by a unique recombinational single step deletion process termed telomere rapid deletion (TRD). TRD involves invasion of the elongated 3' G tail into the proximal telomeric tract resulting in the formation of the D loop structure. Following branch migration the D-loop is nicked and resolved into a deleted telomere and a circular liner product. Cells deleted for MRE11, RAD50 or XRS2 are deficient in TRD process. It has been hypothesized that Mre11 could be a candidate for cleaving the D-loop structure. The endonuclease activity of Mre11 on D-loop structure, preferentially at the 5' ends at TG residues demonstrated in this study, show that Mre11 could function as the nuclease required to generate the deleted telomere in TRD. MRX complex is involved in several processes involving DNA metabolism. It is important that the activities of the complex are regulated in the in vivo context. Complex formation and the interaction of the individual subunits with nucleotide cofactors and metal ions constitute a mode of regulation. This study shows that Rad50 regulates the endonuclease, but not exonuclease activity of Mre11. The binding of ATP and its hydrolysis by Rad50 brings in the regulatory factor necessary to keep the uncontrolled nuclease activity of MRX in check, thus preventing any deleterious effects on telomere length. Telomere maintenance by telomerase is activated in 80% of cancer cells. Inhibition of telomerase by G quartets provides a new drug targets for potential anti-cancer drugs. It is, therefore, likely that understanding the biological consequences of G quadruplex interactions would provide a better insight in development of therapeutics for cancer.

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