Universiẗat, Diss., 2004--Köln.

La protéine télomérique TRF2 permet de protéger les télomères notamment en régulant leur taille. Dans des cellules humaines, la surexpression de la protéine mutante TRF2ΔB, dont le domaine basique est absent, induit un raccourcissement soudain des télomères. In vitro, ce domaine basique protège des structures d’ADN particulières, appelées Jonctions de Holliday (JH), de la résolution par des endonucléases. Ces JH peuvent être présentes aux télomères d’une part au niveau de la boucle télomérique, une conformation de l’ADN qui ressemble à une structure intermédiaire de la recombinaison homologue (RH), et d’autre part au niveau des fourches de réplication bloquées, fréquentes aux télomères. Nous pensons que le raccourcissement soudain des télomères implique la résolution de JH au cours d’un événement de recombinaison homologue qui doit être étroitement régulé afin d’éviter qu’il ne se réalise de façon inappropriée. Dans le but de mieux caractériser cet événement, j’ai montré que différentes endonucléases capables de résoudre des JH (GEN1, MUS81, SLX1-SLX4) sont impliquées dans le raccourcissement des télomères induit par la surexpression de la protéine TRF2ΔB. Puis j’ai étudié le rôle de la protéine hRAP1 dans la régulation de ce mécanisme et l’implication des protéines de la RH. L’ensemble des résultats obtenus nous ont permis de proposer un nouveau rôle de la protéine TRF2 dans la régulation des événements de recombinaison homologue au cours de la réplication des télomères. / The stability of mammalian telomeres depends upon TRF2 which prevents inappropriate repair and checkpoint activation. In human cells, overexpressing a TRF2 mutant lacking the N-terminal basic domain, TRF2ΔB, induces sudden telomere shortening. In vitro, the basic domain protects particular DNA structures, called Holliday junctions (HJ), of the resolution by endonucleases. These HJ may be present at telomeres in one hand at the t-loop, a DNA conformation looking like a structural intermediate of homologous recombination (HR), and also at the level of stalled replication forks, frequent at telomeres. We believe that the sudden shortening of telomeres involves the resolution of HJ during a HR event that would be tightly regulated to prevent it occurs inappropriately. In order to better characterize this event, I have shown that different proteins harbouring resolving activities (GEN1, MUS81, SLX1-SLX4) are involved in telomere shortening induced by overexpression of TRF2ΔB. Then, I studied the role of hRAP1 in the regulation of this mechanism and involvement of HR proteins. The overall results allowed us to propose a new role of TRF2 in the regulation of HR events during the replication of telomeres.

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23091号 / 医博第4718号 / 新制||医||1050(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 篠原 隆司, 教授 増永 慎一郎, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Hoosier born John Hampden Holliday, Civil War soldier, publisher, and civic benefactor grew to manhood in the city of his birth, Indianapolis, and there came to be recognized as one of the foremost pioneer journalists of his time.

Saccharomyces cerevisiae HOP1, which encodes a component of the synaptonemal complex, plays an important role in both gene conversion and crossing over between homologs, as well as enforces the meiotic recombination checkpoint control over the progression of recombination intermediates. The zinc-finger motif (Znf) 348CX2CX19CX2C374) of Hop1 is crucial for its function in meiosis, since mutation of conserved Cys371 to Ser in this motif results in a temperature-sensitive phenotype, which is defective in sporulation and meiosis. The direct role for Hop1 or its ZnF in the formation of joint molecules and checkpoint control over the progression of meiotic recombination intermediates is unknown. To understand the underlying biochemical mechanism, we constructed a series of recombination intermediates. Hop1 or its ZnF were able to bind different recombination intermediates. Interestingly, the binding affinity of Hop1 and its ZnF was much higher for the Holliday junction as compared to other recombination intermediates. The complexes of Hop1 or its ZnF with the Holliday junction were stable and specific as shown by NaCl titration and competition experiment. Hop1 and its ZnF blocked BLM helicase-induced unwinding of the Holliday junction, indicating that the interaction between Hop1 and its ZnF with the Holliday junction is specific. DNase I footprinting experiment showed that Hop1 or its ZnF bind to the center of the Holliday junction. 2-aminopurine fluorescence and KMnO4 experiments showed that Hop1 or its ZnF can distort the Holliday junction in a 2-fold symmetrical manner. The molecular modeling study showed that Hop1 ZnF folded into unique helix-loop-helix motif and bound to center of the Holliday junction. In summary, this study shows that Hop1 protein or its ZnF interact specifically with the Holliday junction and distort its structure. Taken together, these results implicate that Hop1 protein might coordinate the physical monitoring of meiotic recombination intermediates during the process of branch migration and that Hop1 ZnF acts as a structural determinant of Hop1 protein functions.

Homologous recombination (HR) is conserved across all three domains of life and is associated with a number of key biological processes. Over the years, numerous genetic, biochemical and structural studies have uncovered important mechanistic details and established a role for HR in DNA damage repair, control of DNA replication fidelity and suppression of various types of cancer. Much of our current understanding of the mechanistic aspects of HR is gained from the study of Escherichia coli paradigm. E. coli RecA is the founding member of a nearly ubiquitous family of multifunctional proteins and is substantially conserved among eubacterial species. During HR, RecA protein promotes homologous pairing followed by strand exchange reaction leading to heteroduplex formation. In addition to HR, RecA is a central component of SOS response, recombinational DNA repair and rescue of collapsed replications forks. Moreover, recent work has suggested that DNA recombination/repair mechanisms might contribute to genome evolution and consequently to the generation of multidrug-resistant strains of the pathogen. The disease caused by Mycobacterium tuberculosis, endemic in certain regions of the world, is a leading cause of disability and death. A thorough knowledge of the function and interaction of specific HR proteins/enzymes involved in the maintenance of genome integrity is essential in order to elucidate the impact of genome perturbation effects on M. tuberculosis. Toward this end, modulation of RecA protein activity, a central component of HR, represents a potential novel target for design of new drugs because of its involvement in various processes of DNA metabolism. Additionally, small molecule modulators of RecA activity may offer novel insights into the regulation and its role in cellular physiology and pathology. Traditionally, antibiotics have been used to treat infections caused by bacteria. Despite their importance, the development of new antibiotics against M. tuberculosis has considerably decreased over the past several years due to disappointing results in clinical trials. These failures may be due the fact that they suffer from low potency or low cell permeability. Therefore, one of the aims of studies described in this thesis was to test the effect of suramin, a known inhibitor of E. coli RecA, on various biochemical activities of mycobacterial RecA proteins and determine its mechanism of action. Furthermore, the most crucial step in the HR pathway and rescue of collapsed DNA replication forks is the resolution of Holliday junctions and other branched intermediates. Because Holliday junction resolvases are essential for the resolution of different types of DNA recombination/repair intermediates, therefore, we considered it worthwhile to study the genomic expression and biochemical properties of HJRs in M. tuberculosis. Suramin is a commonly used antitrypanosomal and antifiliarial drug, and a novel experimental agent for the treatment of several cancers. A forward chemical screen assay identified several small molecule inhibitors of E. coli RecA. In this screen, suramin (also called germanin), a polysulfonated naphthylurea, and suramin-like agents were found to inhibit EcRecA catalyzed ATPase and DNA strand exchange activity. However, the mechanism underlying such inhibitory action of suramin and whether it can exert antibacterial activity under in vivo conditions remains largely unknown. In an attempt to delineate the range of suramin action, we reasoned that it might be useful to test its effect on mycobacterium RecA proteins. We found that suramin is a potent inhibitor of all known biochemical activities of mycobacterial RecA proteins with IC50 values in the low μM range. The mechanism of action involves, in part, its ability to disassemble the nucleoprotein filaments of RecA-ssDNA. To validate the above results and to obtain quantitative data, a pull-down assay was developed to assess the effect of suramin on RecA–ssDNA filaments. The data indicated that suramin was able to dissociate >80% of RecA bound to ssDNA. Altogether, these results indicated the effectiveness of suramin in the disassembly of RecA nucleoprotein filament. Next, we sought to test whether suramin binds to RecA by using a CD spectropolarimeter. Significant spectral changes were observed upon addition of increasing concentrations of suramin, indicating alterations in the secondary structure of RecA protein. Additional evidence revealed that suramin impaired RecA catalyzed proteolytic cleavage of LexA repressor and blocked ciprofloxacin-inducible recA gene expression and SOS response. More importantly, suramin potentiated the cidal action of ciprofloxacin and reduced the growth of Mycobacterium smegmatis recA+ strain but not its isogenic recA∆ mutant, consistent with the idea that it acts directly on RecA protein. This approach, which appears as an appealing concept, opens up new possibilities to chemically disrupt the pathways controlled by RecA and treat drug-sensitive as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies. The annotated genome sequence of M. tuberculosis revealed the presence of putative homologues of E. coli DNA recombination/repair genes. However, it is unknown whether these putative genes have the ability to encode catalytically active proteins or participate in biochemical reactions intrinsic to the process of HR or DNA repair. Studies in the second half of the thesis originated from an in silico analysis for genes that encode functional equivalents of E. coli RuvC HJ resolvase(s) in M. tuberculosis. The central intermediate formed during mitotic and meiotic recombination is a four-way DNA junction, also known as the Holliday junction (HJ), and its efficient resolution is essential for proper segregation of chromosomes. The resolution of HJ is mediated by a group of structure specific endonucleases known as the Holliday junction resolvases (HJR) which have been identified in a wide variety of organisms based on their shared biochemical characteristics. Bioinformatics analyses of the evolutionary relationships among HJ resolvases suggests that HJR function has arisen independently from four distinct structural folds, namely RNase H, endonuclease VII-colicin E, endonuclease and RusA. Furthermore, similar analyses of HJRs identified another family within the RNaseH fold, along with previously characterized RuvC family of junction resolvases. This new family of putative HJRs is typified by E. coli Yqgf protein. The yqgf gene is highly conserved among bacterial genomes. Nuclear magnetic resonance structural studies have disclosed notable structural similarities between E. coli RuvC and YqgF proteins. Utilizing homology-based molecular modelling, YqgF is predicted to function as a nuclease in various aspects of nucleic acid metabolism. Sequence analysis of M. tuberculosis genome has revealed the presence of two putative HJ resolvases, ruvC (Rv2594c) and ruvX (Rv2554c, yqgF homolog). Previous studies have demonstrated that M. tuberculosis ruvC is induced following DNA damage and ruvX is expressed during active growth phase of M. tuberculosis. More importantly, the absence of ruvC increased the potency of moxifloxacin in M. smegmatis. Although, these results imply that the ruv genes play crucial roles in DNA recombination and repair in M. tuberculosis, the biochemical properties of their gene products have not been characterized. In this study, we have isolated M. tuberculosis ruvC and yqgF genes and purified their encoded proteins, M. tuberculosis RuvC (MtRuvC) and M. tuberculosis RuvX (MtRuvX), respectively, to near homogeneity. Protein-DNA interaction assays conducted with purified MtRuvC and MtRuvX revealed that both can bind HJ, albeit with different affinities. However, in contrast to MtRuvC, MtRuvX showed robust HJ resolvase activity. The endonuclease activity of MtRuvX was completely dependent on Mg2+and Mn2+ partially substituted for Mg2+. Additional experiments showed that RuvX exhibits >2-fold higher binding affinity for HJ over other recombination/ replication intermediates. As demonstrated for other HJRs, MtRuvX failed to cleave static HJ and linear duplex DNA. The cleavage sites were mapped within the homologous core of a branch-migratable HJ. To identify catalytic residues in RuvX, we conducted mutational analysis of an acidic amino acid residue guided by the bioinformatics data. The product of MtRuvXD28N retained full HJ-binding activity, but showed extremely reduced HJ-specific endonuclease activity. Further biochemical characterization revealed that MtRuvX exists as a homodimer in solution. Notably, we found that disulfide-bond mediated intermolecular homodimerization is crucial for the ability of MtRuvX to cleave Holliday junctions, implicating that stable junction binding is necessary to promote branch migration and to create cleavable sites. Analysis of qPCR data suggested that the pattern of yqgF gene expression was similar to those of ruvC and recA genes following DNA damage. Together, these data indicate that ruvX expression is induced by DNA-damaging agents and that RuvX might be functionally involved in recombinational DNA repair in M. tuberculosis. These findings are all consistent with the idea that RuvX might be the bona fide HJ resolvase in M. tuberculosis analogous to that of E. coli RuvC. More importantly, we provide the first detailed characterization of RuvX and present important insights into the mechanism of HJ resolution, which could be directly linked to the regulation of different DNA metabolic processes, including HR, DNA replication and DNA repair. Overall, this study opens a new avenue in the understanding of HR in this human pathogen, together with elucidation of the function of some of the uncharacterized genes may represent a novel set of recombination enzymes.

Par micromanipulation et mesure de force sur molécule unique, avec un piège magnétique, ce travail a porté en partie sur l'étude du système de réparation « à longue distance » de l'ADN. Cette réparation fait intervenir pour son initiation les protéines MutS, MutL, et MutH et utilise un mécanisme non identifié précisément, qui lui permet<br />d'agir à distance, entre un site de mésappariement de l'ADN (dû par exemple à une erreur de réplication), et un site proximal distant (hémi-méthylation de séquence GATC), ce qui permet de diriger la réparation sur le brin néosynthétisé. Certains modèles de l'action de la protéine MutS font intervenir une boucle dans l'ADN. Nous<br />avons cherché à mettre en évidence une telle action sur un ADN double brin, contenant (ou ne contenant pas) un mésappariement. Nous n'avons pas mis en évidence de<br />formation de boucle par MutS, qui soit spécifiquement liée à la présence d'un mésappariement. Ce résultat négatif semble donc exclure ce modèle de boucle spécifique. Dans une deuxième partie, nous avons effectué des expériences de micromanipulation sur une jonction de Holliday (ADN en forme de croix, intermédiaire de recombinaison). Nous avons montré directement qu'il est possible d'extruder une jonction de Holliday, en sous-enroulant mécaniquement une molécule d'ADN comportant une séquence palindromique, et avons aussi déduit de ces expériences une mesure du pas de l'hélice de l'ADN. Dans une dernière partie, nous avons étudié l'influence du bromure d'éthidium sur l'ADN. Nous avons montré que la présence de cet agent intercalant peut induire une attraction non-spécifique, intra- ou inter- simple brins d'ADN.

Ce travail présente tout d'abord l'étude, à l'échelle de la molécule individuelle, de l'intermédiaire<br />de recombinaison formé par l'échange de simples brins entre deux molécules d'ADN homologues : la<br />jonction de Holliday.<br />Nous montrons tout d'abord qu'il est possible, à partir d'un ADN portant une séquence entièrement<br />palindromique, de former une jonction de Holliday en appliquant une torsion négative. Une fois<br />la jonction formée, la torsion permet également de contrôler de façon directe l'échange des simples brins.<br />Cette technique nous a permis d'accéder expérimentalement, avec une très bonne précision, à la valeur<br />en solution du pas hélicoïdal de l'ADN : 3.61 ± 0.03 nm/tr.<br />Ensuite nous avons étudié, en présence d'ions magnésium, la cinétique de migration de la jonction<br />de Holliday sous l'influence des contraintes mécaniques. Une modélisation simple du comportement<br />de la jonction de Holliday vis-à-vis des contraintes mécaniques a été développée permettant d'expliquer<br />leur influence sur le mécanisme de migration.<br />L'échange des simples brins peut également être catalysé par certaines enzymes. Le travail<br />mécanique développé au cours de cette activité catalytique fait de ces enzymes des moteurs moléculaires.<br />La seconde partie de ce travail porte sur l'étude en molécule unique d'un tel moteur : le complexe RuvAB<br />de la bactérie Escherichia coli.<br />Nous avons tout d'abord caractérisé la migration de jonctions de Holliday individuelles sous<br />l'action du complexe RuvAB. Nous avons notamment montré la très grande processivité du complexe et<br />nous avons pu estimer la vitesse de migration à 37◦C et en présence d'1 mM d'ATP : ∼ 43 paires de<br />bases échangées par seconde.<br />D'autre part, et pour finir, nous avons mis en évidence le rôle catalytique de la sous-unité RuvA<br />dans l'échange des paires de bases au niveau du point de branchement.

La protéine TRF2 est une protéine clé dans la dynamique des télomères, ces structures nucléoprotéiques présentes à l'extrémité des chromosomes linéaires et responsables de leur protection. Bien que la manière dont les télomères s'organisent pour protéger l'ADN soit encore méconnue, il a été montré récemment que TRF2 est à l'origine de la formation d'une structure en boucle, ou boucle télomérique qui empêcherait les extrémités télomériques d'être reconnus comme des coupures double brin. Un modèle propose que TRF2 permette la formation de cette boucle en induisant l'invasion du simple brin télomérique terminal à l'intérieur de la séquence double brin après repliement du télomère sur lui même. Les études réalisées lors de cette thèse montrent que TRF2 est en mesure de stimuler l'invasion télomérique de manière indirecte facilitant l'ouverture de la double hélice grâce à des modifications d'ordre topologique de l'ADN cible. Par ailleurs, les travaux réalisés mettent également en évidence un second mode de fixation à l'ADN de TRF2, par l'intermédiaire de son domaine N-terminal qui possède une affinité remarquable pour la structure des jonctions de Holliday. La dernière partie de cette thèse met en évidence l'activité 5' exonucléase d'une nouvelle protéine télomérique, la protéine Apollon, qui serait impliquée dans la protection des télomères. Tous ces résultats participent à une meilleure compréhension du fonctionnement de TRF2 sur les télomères et en particulier de son rôle dans la formation de la boucle télomérique.

The goal of the creative project was to provide recommendations for renovating the Holliday Park Arboretum based upon existing conditions, tree species, open space analysis and interpretive activities.All trees within the Holliday Park Arboretum were mapped, tagged and identified by species. An Indiana native tree species list was developed and compared to the existing tree species in the arboretum. It was then determined how many native and exotic trees existed in the arboretum. Native tree species not existing in the arboretum were placed on a separate list to establish which native trees were needed to have a representative of each native tree in the arboretum.An open space analysis was conducted to determine potential planting areas within the arboretum and the number of additional native trees that could be planted in those spaces. Recommendations were provided for the selection and planting of native trees as part of the renovation process that can be used by the park administration. / Department of Landscape Architecture