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Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

<p> Mre11 and Rad50 form a complex with Nbs1 (MRN) in mammals and Xrs2 (MRX) in yeast. The MRN complex plays a role in many cellular processes, such as DNA damage sensing, DNA repair, cell cycle checkpoint and telomere maintenance. Rad50 contains a conserved ATP binding motif and its ATPase activity is essential for ATM activation in vitro. Using a tethering approach, I have shown that Rad50 can be targeted to telomeres through its fusion to hRap1. The fusion of hRap1 to Rad50 did not alter the property of Rad50. The fused wild-type Rad50 promoted telomerase-dependent telomere lengthening. However, the fusion proteins containing loss-of-function mutations in Rad50 (K42E and S1202R) did not. I have also shown that the fused wild-type Rad50 was able to form irradiation-induced foci in a manner similar to unfused Rad50. In contrast, the two defective mutants of Rad50 failed to accumulate irradiation-induced foci. Expression of the fusion proteins containing Rad50 mutants also interfered with the ability of endogenous Mre11 protein to form foci post irradiation. Thus our data suggest that the Rad50 mutants may function as dominant-negative alleles in cells.</p> / Thesis / Master of Science (MSc)

DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.

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.

Eukaryotic cells repair DNA double-strand breaks (DSBs) through both non-homologous and homologous recombination pathways. The initiation of homologous recombination requires the generation of 3' overhangs, which are essential for the formation of Rad51 protein-DNA filaments that catalyze subsequent steps of strand invasion. Experiments in budding yeast show that resection of the 5' strand at a DSB is delayed in strains lacking any components of the Mre11/Rad50/Xrs2 (MRX) complex¹ . In meiosis, a specific class of hypomorphic mutants of mre11 and rad50 (Rad50S) are completely deficient in 5' resection and leave Spo11 covalently attached to the 5' strands of DNA breaks². Similar to mre11S and rad50S mutants, sae2 deletion strains fail to resect 5' strands at meiotic DSBs and accumulate covalent Spo11 adducts³;⁴. In addition, Sae2 and MRX were also found to function cooperatively to process hairpin-capped DNA ends in vivo in yeast. sae2 and mrx null strains show a severe defect in processing these structures and accumulate hairpin-capped DNA ends⁵;⁶. The Longhese laboratory has also shown that Sae2 deletion strains show a delay in 5' strand resection, similar to rad50S strains⁷. Recently, Bettina Lengsfeld in our laboratory demonstrated that Sae2 itself possesses nuclease activity and that MRX and Sae2 act cooperatively to cleave single-stranded DNA adjacent to DNA hairpin structures⁸. In vitro characterization of Sae2 showed that the central and N-terminal domains are required for MRX-independent nuclease activity and that the C-terminus is required for cooperative activities with MRX. Sae2 also acts independently of MRX as a 5' flap endonuclease on branched structures in vitro. Our studies investigate whether MRX, Sae2, and Exo1 function cooperatively in DNA resection using recombinant, purified proteins in vitro. We developed assays utilizing strand-specific Southern blot analysis to visualize DNA end processing of model DNA substrates using recombinant proteins in vitro. Our results demonstrate that MRX and Sae2 cooperatively resect the 5' end of a DNA duplex together with the Exo1 enzyme, supporting a role for these factors in the early stages of homologous recombination and repair. / text

Abstract Mutations in BRCA1 and BRCA2 explain only about 20% of familial aggregation of breast cancer, suggesting involvement of additional susceptibility genes. In this study five DNA damage response genes, ATM, ATR, MRE11, NBS1 and RAD50, were considered as putative candidates to explain some of the remaining familial breast cancer risk, and were screened for germline mutations in families displaying genetic predisposition. Analysis of ATM indicated that clearly pathogenic mutations seem to be restricted to those reported in ataxia-telangiectasia (A-T). However, a cancer risk modifying effect was suggested for a combination of two ATM polymorphisms, 5557G>A and IVS38-8T>C, as this allele seemed to associate with bilateral breast cancer (OR 10.2, 95% CI 3.1–33.8, p = 0.001). The relevance of ATM mutations, originally identified in Finnish A-T patients, in breast cancer susceptibility was evaluated by a large case-control study. Two such alleles, 6903insA and 7570G>C, in addition to 8734A>G previously associated with breast cancer susceptibility, were observed. The overall mutation frequency in unselected cases (7/1124) was higher than in controls (1/1107), but a significantly elevated frequency was observed only in familial cases (6/541, p = 0.006, OR 12.4, 95% CI 1.5–103.3). These three mutations showed founder effects in their geographical occurrence, and had different functional consequences at protein level. In ATR no disease-related mutations were observed, suggesting that it is not a breast cancer susceptibility gene. The mutation screening of the Mre11 complex genes, MRE11, NBS1 and RAD50, revealed two novel potentially breast cancer associated alleles: NBS1 Leu150Phe and RAD50 687delT were observed in 2.0% (3/151) of the studied families. The subsequent study of newly diagnosed, unselected breast cancer cases indicated that RAD50 687delT is a relatively common low-penetrance susceptibility allele in Northern Finland (cases 8/317 vs. controls 6/1000, OR 4.3, 95% CI 1.5–12.5, p = 0.008). NBS1 Leu150Phe (2/317) together with a novel RAD50 IVS3-1G>A mutation (1/317) was also observed, both being absent from controls. Loss of the wild-type allele was not observed in the tumors of the studied mutation carriers, but they all showed an increase in chromosomal instability of peripheral T-lymphocytes. This suggests an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity and susceptibility to cancer.

No description available.

Le parvovirus humain Adeno-Associé (AAV) est un Dependoparvovirus qui ne peut accomplir son cycle réplicatif qu’en présence d’un virus auxiliaire tel que l’Adénovirus (AdV) ou le virus de l’Herpès Simplex de type 1 (HSV-1). En absence de virus auxiliaire, l’AAV va persister sous forme épisomale ou intégrée. Cette intégration survient de façon préférentielle dans un locus spécifique, au site AAVS1, présent sur le chromosome 19 du génome humain.Des travaux précédents ont porté sur l’étude du contrôle de la réplication de l’AAV par les facteurs cellulaires de réparation des cassures d’ADN. En particulier, le complexe MRN (Mre11/Rad50/Nbs1), un senseur majeur des cassures de l’ADN double brin (CDB), a été montré comme pouvant inhiber les réplications virales de l’AAV et de l’AdV lors d’une co-Infection. L’AdV est capable de contrer cet effet en induisant la délocalisation et la dégradation de MRN. A l’opposé, MRN participe de façon positive à la réplication de l’HSV-1 et se retrouve localisé dans les centres de réplication viraux (CR) de l’AAV induits par HSV-1. Ceci nous a conduits à explorer plus en détail le rôle de ce complexe sur la réplication de l’AAV en présence d’HSV-1. Les résultats obtenus indiquent, qu’en absence de MRN, la réplication du génome de l’AAV est réduite de façon significative dans des cellules co-Infectées avec le virus HSV-1, sauvage ou muté pour son activité polymérase. Cette diminution est spécifique à l’AAV sauvage car aucune perturbation n’est observée sur la réplication des vecteurs AAV recombinants lorsque MRN est absent. La régulation positive de la réplication de l’AAV par MRN est dépendante de l’activité de pontage de l’ADN exercée par Rad50. De façon intéressante, l’absence de MRN inhibe également de façon significative l’intégration préférentielle de l’AAV au site AAVS1, que ce soit en absence ou en présence d’HSV-1.Ce travail de thèse suggère que le complexe MRN régulerait de façon différentielle la réplication de l’AAV en fonction du virus auxiliaire qui l’accompagne et identifie, pour la première fois, MRN comme un facteur clé pour l’intégration du génome de l’AAV au site AAVS1. / Adeno-Associated Virus (AAV) is a helper dependent Dependoparvovirus that requires co-Infection with adenovirus (AdV) or herpes simplex virus type 1 (HSV-1) to productively replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Integration occcurs preferentially at a specific locus called AAVS1 and based on human chromosome 19.Previous studies have analyzed the DNA damage response induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DNA damage response induced by double-Stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during co-Infection. AdV counteracts this effect by inducing the delocalization and degradation of MRN. In contrast, MRN favors HSV-1 replication and our previous studies showed that it was recruited to AAV replication compartments that were induced in the presence of HSV-1. In this study we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after co-Infection with polymerase deleted or wild type HSV-1. This reduction was specific of wild type AAV since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering and nuclease activities. Importantly, knockdown of MRN could also negatively regulate AAV site-Specific integration within the human AAVS1 site, an event which occurred at a significant level during AAV replication induced by co-Infection with HSV-1. Altogether, this work demonstrates that MRN can differentially regulate AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during site-Specific integration of the AAV genome.

Les télomères sont des structures nucléoprotéiques spécialisées dont l'un des rôles est d'empêcher le raccourcissement progressif de l'extrémité des chromosomes suite à la réplication et à l'instabilité génomique due à la recombinaison de l'extrémité de chromosomes. Malgré le rôle des télomères dans la protection de l'extrémité des chromosomes contre les mécanismes de réparation de l'ADN et de recombinaison, de nombreuses protéines de ces voies jouent des rôles essentiels dans l'homéostasie télomérique et la stabilité des chromosomes. Parmi elles, la protéine RAD50 appartenant au complexe MRE11/RAD50/XRS25(NBS1) et l'endonucléase structure spécifique XPF/ERCC1 sont localisées aux télomères ; ces deux complexes connus pour leur rôle dans les voies de réparation de l'ADN ainsi que dans les études sur la recombinaison. Nous avons identifié deux rôles différents pour la protéine RAD50 dans la maintenance télomérique et dans la protection des extrémités des chromosomes, en contexte de présence et absence de la télomérase. L'absence d'AtRAD50 augmente significativement le nombre de fusions chromosomiques impliquant des télomères raccourcis. Nous proposons que ce rôle protecteur des télomères raccourcis de RAD50 est le résultat de sa fonction de contraindre la recombinaison entre chromatides soeurs et ainsi d'éviter les évènements de fusions par les extrémités. Nous avons recherché le ou des mécanismes impliqué(s) dans ces évènements de fusions chromosomiques chez les mutants atrad50 en réalisant des croisements entre les plantes déficientes pour ATRAD50 et des plantes déficientes pour des gènes codant des protéines des voies de réparation par recombinaison non-homologue et homologue. Au contraire de la situation en cellules de mammifères, nous n'avons pas observé d'instabilité chromosomique chez les plantes mutantes correspondantes pour XPF (AtRAD1) or ERCC1 (AtERCC1). Cependant, en absence de la télomérase, la mutation de l'un de ces deux gènes entraîne une augmentation précose et significative de l'instabilité chromosomique sans accélération générale de la perte des répétitions télomériques, mais associée à la présence de fragments ADN extrachromatiques visibles en cytologie. Une analyse intensive par FISH a permis d'identifier ces ADN comme des bras entiers spécifiques de deux chromosomes. Nos données indiquent un rôle protecteur de RAD1/ERCC1 comme l'invasion de l'ADN simple brin télométrique dans des séquences télomériques interstitielles. Le fait que les mutations de rad1 (ou ercc1) augmentent dramatiquement l'instabilité chromosomique des mutants télomérase a des implications très importantes pour les modèles des rôles de la recombinaison aux télomères.