Tesis sobre el tema "Recombinase RecA"

Homologous recombination is an essential pathway in the repair of DNA damage during the DNA replication process. RecA protein promotes the central steps in homologous recombination, after coating single-stranded DNA (ssDNA), RecA carries out a pairing and strand exchange reaction involving homologous DNA. This research project aims to characterize RecA function in homologous recombination using single molecule tethered particle motion (TPM). Using TPM to observe RecA extension along DNA, the RecA extension rate on ssDNA was determined for the first time. The rate obtained for dsDNA was similar, implying that RecA polymerizes along only one strand of a DNA substrate. The nucleation behaviour of RecA on DNA was also obtained from the extension trace, confirming the hypothesis that rapid nucleation on ssDNA is pH independent, while nucleation on dsDNA is pH dependent. Several pilot single molecule experiments aimed at monitoring the pairing and strand exchange reaction in real time were attempted. Although these experiments were unsuccessful, successful ensemble biochemical analogues of these experiments proved the feasibility of the single molecule experiments. These attempts gave insights into possible factors hindering success and led to experimental suggestions essential to the success of future experiments.
La recombinaison Homologue est un chemin essentiel dans la réparation de dommages d'ADN pendant le procédé de réplication d'ADN. La protéine de RecA promeut les étapes centrales dans la recombinaison homologue, après avoir revêtu ADN seul-abandonné (ssDNA), RecA exécute un mettre et la réaction d'échange de brin impliquant ADN homologue. Ce projet de recherche vise à caractériser la fonction de RecA dans la recombinaison homologue utilisant la molécule seule mouvement de particule attaché (TPM). TPM d'utilisation pour observer l'extension de RecA le long d'ADN, le taux d'extension de RecA sur ssDNA a été déterminé pour la première fois. Le taux obtenu pour dsDNA était similaire, impliquant ce RecA polymerizes le long de seulement un brin d'un substrat d'ADN. Le comportement de nucleation de RecA sur ADN a été aussi obtenu de la trace d'extension, confirmant l'hypothèse ce nucleation rapide sur ssDNA est indépendant du pH, pendant que nucleation sur dsDNA est dépendant du pH. Plusieurs pilote plusieurs expériences de molécule seules ont visé à contrôlant le mettre et la réaction d'échange de brin a été tentée en temps réel. Bien que ces expériences étaient les ensembles infructueuses et réussies analogues biochimiques de ces expériences ont prouvé la possibilité des expériences de molécule seules. Ces tentatives ont donné de l'aux perspicacités dans les facteurs possibles freinant le succès et a mené à l'élément essentiel de suggestions expérimental au succès d'expériences futures

La transformation naturelle est la capacité de certaines bactéries à incorporer et à intégrer activement de l’ADN extra-cellulaire. Ce procédé majeur augmente la plasticité et l’adaptabilité des bactéries à Gram positif et négatif en réalisant des échanges génétiques intra- et inter-espèces. S. pneumoniae est un pathogène majeur de l’Homme et se retrouve de façon commensale dans les muqueuses du nasopharynx. Cette bactérie est responsable d’infections sévères telles que des pneumonies, des méningites et des septicémies. Dans cette espèce, la transformation naturelle est corrélée au phénomène de changement de capsule et à la baisse d’efficacité des vaccins, ainsi qu’à l’obtention de gènes de résistance aux antibiotiques. Au cours de la transformation, l’ADN extra-cellulaire va être pris en charge dans le cytoplasme par différentes protéines, qui permettront à terme d’intégrer ce gène au génome bactérien par recombinaison homologue. La recherche d’homologie et l’intégration du gène est sous le contrôle de la recombinase universelle RecA. Pour cela, RecA va former un filament hélicoïdal avec l’ADN sb exogène et db endogène, et ceux de manière ATP dépendante. Toutefois, la structure atomique de tels filaments n’a jamais été observé chez S. pneumoniae. Par des techniques de caractérisation biochimique et de cryo-microscopie électronique nous sommes parvenus à résoudre la structure des deux types de filaments à des résolutions de 3.8 Å et 3.9 Å. Ce qui nous a permis par la suite de mieux caractériser l’interaction de RecA avec l’ADN. En comparant nos structures avec celles obtenues par cristallographie des filaments de RecA observé chez E. coli, nous avons en partie expliqué les raisons de l’efficacité de recombinaison 3 fois supérieur de S. pneumoniae. Dans un second temps, nous avons mis en évidence in vitro un lien entre la filamentation de RecA le long de l’ADN et l’activité hélicase de RadA. RadA est une protéine nécessaire à la recombinaison homologue et dont l’activité hélicase semble promouvoir et étendre la D-loop au niveau de l’ADN db endogène. Nous avons également caractérisé, par RMN, la structure du motif en doigt de zinc de RadA et son interaction avec l’ADN, motif qui n’avait pu être résolu dans la structure de RadA en cristallographie et qui semble indispensable à son activité hélicase
Natural transformation is the ability of some bacteria to incorporate and actively integrate extracellular DNA. This major process increases the plasticity and adaptability of gram positive and negative bacteria by performing intra- and inter-species genetic exchanges. S. pneumoniae is a major human pathogen and is found commensally in the mucous membranes of the nasopharynx. This bacterium is responsible for severe infections such as pneumonia, meningitis and sepsis. In this species, the natural transformation is correlated with the phenomenon of capsule change and the decrease of vaccine efficacy, as well as the obtaining of genes for antibiotic resistance. During the transformation, the extracellular DNA will be supported in the cytoplasm by different proteins, which will eventually integrate this gene into the bacterial genome by homologous recombination. The search for homology and the integration of the gene is under the control of the universal recombinase RecA. For this, RecA will form a helical filament with exogenous ssDNA and endogenous dsDNA, and those in ATP dependent manner. However, the atomic structure of such filaments has never been observed in S. pneumoniae. Using biochemical characterization techniques and electron cryomicroscopy, we succeeded in solving the structure of the two types of filaments at resolutions of 3.8 Å and 3.9 Å. This allowed us to better characterize the interaction of RecA with DNA. By comparing our structures with those obtained by crystallography of RecA filaments observed in E. coli, we partly explained the reasons for the 3-fold recombination efficiency of S. pneumoniae. In a second step, we demonstrated in vitro a link between the RecA filamentation along the DNA and the helicase activity of RadA. RadA is a protein necessary for homologous recombination and whose helicase activity seems to promote and extend D-loop at the level of endogenous dsDNA. We have also characterized, by NMR, the structure of the zinc finger motif of RadA and its interaction with DNA, a motif that could not be solved in the RadA structure in crystallography and which seems essential for its helicase activity

Bacillus subtilis is the model organism for low GC Gram-positive bacteria and is of great biotechnological interest. Protein phosphorylation is an important regulatory mechanism in bacteria and it has not been extensively studied yet. Recent site-specific phosphoproteomic studies identified a large number of novel serine/threonine phosphorylation sites in B. subtilis, including a) two transition phase global gene regulators DegS and AbrB and b) RecA, that plays a major role in double-strand break repair and DNA recombination. .B. subtilis disposes of several putative Ser/Thr kinases like PrkA, YbdM, YabT and a characterizd kinase PrkC, but very few physiological substrates for these have been defined so far. In vitro phosphorylation assays were used to identify which of these kinases were able to phosphorylate DegS, RecA and AbrB. DegS phosphorylation on serine 76 by the kinase YbdM influenced its activity towards DegU both in vitro and in vivo, and expression of DegS S76D( on replacing serine to aspartate) in B. subtilis perturbed cellular processes regulated by the DegS/DegU two component system. This suggests a link between DegS phosphorylation at serine 76 and the level of DegU phosphorylation, establishing this post-translational modification as an additional trigger for this two-component system. At the onset of sporulation, B. subtilis expresses an unusual serine/threonine kinase YabT, which exhibits a septal localization and is activated by non-sequence-specific DNA binding. Activated YabT phosphorylates RecA at the residue serine 2, which in turn promotes the formation of RecA foci at the onset of spore development. On the other hand, non-phosphorylatable RecA or inactivated YabT lead to reduced spore formation in the presence of DNA lesions . This suggests a functional similarity between B. subtilis developmental stage dependent RecA phosphorylation and its eukaryal homologous Rad51 phosphorylation, which leads to its recruitment to the lesion sites. We therefore proposed that RecA phosphorylation serves as an additional signal mechanism that promotes focus formation during spore development. AbrB is phosphorylated by YabT, YbdM and PrkC in vitro and AbrB phosphorylation leads to reduced affinity for its target DNA and abolished binding cooperativity in vitro and in vivo. Expression of the phosphomimetic AbrB-S86D or of the non-phosphorylatable AbrB-S86A mutant protein in B. subtilis disturbed some stationary phase phenomena such as exoprotease production, competence and the onset of sporulation, probably by deregulation of AbrB-target genes and operons. We therefore, proposed that AbrB phosphorylation as an additional regulatory mechanism needed to switch off this ambiactive gene regulator during the transition phase.

Le génome de Deinococcus deserti, une bactérie très radiotolérante, a été analysé et comparé à ceux de D. Radiodurans et D. Geothermalis. Environ 230 protéines sont spécifiquement conservées chez ces 3 espèces, dont IrrE, un régulateur essentiel pour la radiotolérance. D. Deserti possède plusieurs gènes supplémentaires liés à la réparation de l’ADN, dont imuY et dnaE2 (ADN polymérases translesionnelles). En plus, D. Deserti a 3 recA qui codent pour 2 protéines RecA différentes (RecAC et RecAP). Pour étudier ces gènes, des outils génétiques ont été mis au point. Différents résultats suggèrent qu’IrrE, nécessaire pour l’induction de plusieurs gènes après irradiation, a une activité peptidase. Les 2 RecA sont fonctionnelles pour la réparation de l’ADN. D. Deserti est mutable par UV, ce qui nécessite ImuY, DnaE2 et RecAC, mais pas RecAP
The genome of Deinococcus deserti, a highly radiation-tolerant bacterium, was analyzed and compared to those of D. Radiodurans and D. Geothermalis. About 230 proteins are specifically conserved in these 3 species, including IrrE, a regulator protein essential for radiotolerance. D. Deserti has several supplementary DNA repair genes, like imuY and dnaE2 (translesion DNA polymerases). Moreover, D. Deserti has 3 recA that code for 2 different RecA proteins (RecAC et RecAP). To study these genes, genetic tools were developed for D. Deserti. Different results suggest that IrrE, required for the induction of several genes after irradiation, has peptidase activity. The 2 RecA proteins are functional for DNA repair. D. Deserti is mutable by UV, which requires ImuY, DnaE2 and RecAC, but not RecAP

Homologous genetic recombination occurs during the life cycle of virtually every organism Genetic studies especially in prokaryotes and fungi have defined the rules of recombination, led to the characterization of alternate pathways and to the development of molecular models The biochemistry of homologous genetic recombination has advanced most productively in bacteria and fungi due to the extensive genetic understanding of these organisms The identification of mutants defective in homologous recombination, purification and characterization of the gene products that participate in recombination has brought the ultimate goal of reconstituting a cell-k free system for Eschenchia coli, at least with naked DNA substrates, closer to reality.

Homologous genetic recombination occurs during the life cycle of virtually every organism Genetic studies especially in prokaryotes and fungi have defined the rules of recombination, led to the characterization of alternate pathways and to the development of molecular models The biochemistry of homologous genetic recombination has advanced most productively in bacteria and fungi due to the extensive genetic understanding of these organisms The identification of mutants defective in homologous recombination, purification and characterization of the gene products that participate in recombination has brought the ultimate goal of reconstituting a cell-k free system for Eschenchia coli, at least with naked DNA substrates, closer to reality.

Les protéines jouent un rôle central dans de nombreux processus cellulaire et peuvent intervenir dans de nombreuses interactions différentes, avec d'autres protéines, de l'ADN, des lipides, ou de petits ligands. La détermination de ces interactions est fondamentale pour pouvoir comprendre des processus biologiques majeurs et de nombreuses méthodes expérimentales ont été développées pour les caractériser. Cependant les méthodes expérimentales sont longues et coûteuses et les méthodes informatisées de prédictions d'interactions pourraient, à terme, fournir dans ce contexte une aide précieuse permettant de guider de futures expériences en biochimie et biologie moléculaire. Le développement logiciels d'amarrage est également un processus difficile mettant en jeu des cycles de conception d'algorithme, d'implémentation et de tests. Au cours de ma thèse, j'ai développé une librairie orientée objet pour favoriser et accélérer les étapes d'implémentation et de tests des méthodes d'amarrage. Cette librairie, programmée en C++ et interfacée avec le langage de script Python, a été utilisée pour mettre au point et tester de nouvelles méthodes appliquées à l'amarrage protéine-ADN et à l'amarrage multi-composants. Des programmes développés à l'aide de cette librairie sont actuellement appliqués à l'étude des modes d'amarrage de l'ADN au complexe RecA, responsable de la recombinaison homologue chez les bactéries
Proteins play a central role in various cellular processes with various interactions with other proteins, DNA, lipids or small ligands. Because the determination of these interactions is fundamental for understanding key biological processes, several experimental methods have been developed to characterize them. Experimental studies can take a long time and an expensive. Computational methods can therefore be of great help to guide future biochemical experiments. Development of docking software is a long process involving cycles of algorithm conception, programming and tests. During my thesis, I developed an object oriented library to help and speed-up development and tests of docking methods. This library was programmed in C++ with Python bindings, and has been used to test new methods applied to protein-DNA docking and multicomponent docking. Programs made with the help of this library are presently used to study the binding of DNA to the RecA complex, responsible of homologous recombination

Les protéines reca et rad51, homologue eucaryotique, jouent un rôle crucial dans la conservation de l'intégrité du génome. Elles catalysent la recombinaison homologue. Reca intervient aussi dans le choix du processus de réparation de l'ADN alors que rad51 intervient dans la régulation de l'apoptose. Elles miment in vitro leurs fonctions en interagissant avec l'ADN et d'autres protéines et nécessitent de l'atp. Pour comprendre le mécanisme d'activation de reca par l'atp, j'ai examine l'effet de nucléotides sur l'interaction reca-ADN. Seuls les nucléotides triphosphates dont la base est une adénine stimulent l'interaction reca-ADN alors que les autres nucléotides, sauf utp, dissocient reca-ADN. D'après l'analyse de la structure cristallographique du complexe reca-adp, la base adénine de l'adp est proche du résidu tyr103 de reca. J'ai examine l'effet de remplacement de cette Tyr par d'autres acides amines aromatiques. Les protéines modifiées sont stimulées uniquement par les nucléotides ayant une adénine comme nucleobase, comme pour reca sauvage. Cependant, on observe une activation partielle par le nucléotide ctp de rad51 modifiée en tyr103 par un trp, alors qu'il inhibe l'activité de reca sauvage, confirmant l'importance de ce contact. J'ai aussi montre que le peptide l2 (résidus 195-209) de reca interagit avec l'ADN simple-brin de manière similaire a reca entière avec l'atp. Cette boucle est le centre catalytique de la réaction d'échange de brins d'ADN par reca et interagit avec l'ADN et l'atp. Ce résultat suggère que l'atp active reca via un changement de conformation de la boucle l2. Enfin, j'ai observé que rad51 de xenope interagit avec l'ADN de manière similaire à reca. J'ai examiné l'implication éventuelle de résidu

The error free repair of DNA double strand breaks through the homologous recombinational repair pathway is essential for organisms of all types to sustain life. A detailed structural and mechanistic understanding of this pathway has been the target of intense study since the identification of bacterial recA, the gene whose product is responsible for the catalysis of DNA strand exchange, in 1965. The work presented here began with defining residues that are important for the assembly and stability of the RecA filament, and progressed to the identification of residues critical for the transfer of ATP-mediated allosteric information between subunits in the protein's helical filament structure. My work then evolved to investigate similar mechanistic details concerning the role of ATP in the human RecA homolog, Rad51. Results from non-conservative mutagenesis studies of the N-terminal region of one subunit and the corresponding interacting surface on the neighboring subunit within the RecA protein, led to the identification of residues critical for the formation of the inactive RecA filament but not the active nucleoprotein filament. Through the use of specifically engineered cysteine substitutions we observed an ATP-induced change in the efficiency of cross subunit disulfide bond formation and concluded that the position of residues in this region as defined by the current crystal structure may not accurately reflect the active form of the protein. These ATP induced changes in positioning led to the further investigation of the allosteric mechanism resulting in the identification of residue Phe217 as the key mediator for ATP-induced information transfer from one subunit to the next. In transitioning to investigate homologous mechanisms in the human pathway I designed a system whereby we can now analyze mutant human proteins in human cells. This was accomplished through the use of RNA interference, fluorescent transgenes, confocal microscopy and measurements of DNA repair. In the process of establishing the system, I made the first reported observation of the cellular localization of one of the Rad51 paralogs, Xrcc3, before and after DNA damage. In addition we found that a damage induced reorganization of the protein does not require the presence of Rad51 and the localization to DNA breaks occurs within 10 minutes. In efforts to characterize the role of ATP in human Rad51 mediated homologous repair of double strand breaks we analyzed two mutations in Rad51 specifically affecting ATP hydrolysis, K133A and K133R. Data presented here suggests that, in the case of human cells, ATP hydrolysis and therefore binding, by Rad51 is essential for successful repair of induced damage.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
Chromobacterium violaceum is a ?-proteobacteria commonly found around tropical and subtropical regions throughout the globe. It produces many metabolites with biotechnological properties such as antitumoral peptides, antibiotics and polymers that have potential to replace the oil-based ones. Although it has been extensively studied over the past 40 years, there are many aspects of C. violaceum that remains unclear until today. We have conducted a biochemical study on the homologous recombination (HR) machinery of C. violaceum, mainly in RecA and its paralog, RadA/Sms. We performed in vitro assays from initial and late steps of HR such as D-loop formation and branch migration, respectively, with their corresponding molecular actors and how RadA/Sms influenced each one. We observed cvRadA/Sms influences negatively D-loop formation promoted by cvRecA and through pull-down assay we have observed an interaction between these two proteins. We also observed the DNA-binding preference of cvRadA/Sms and cvRecA and observed that this protein binds preferentially to dsDNA instead ssDNA, unlike cvRecA. No involvement of cvRadA/Sms on branch migration reactions was detected. In this work, we also described, for the first time, the isolation, sequencing and annotation of a new plasmid from C. violaceum, which we named ChVi1 and has 44,236 base pairs, 39 predicted open reading frames (ORFs) and, possibly, two origins of replication. Most of the ORFs codes for hypothetical and structural bacteriophage proteins. By using restriction digestion and Next-generation sequencing (NGS) we also looked for the presence of a similar plasmid in other seven C. violaceum strains isolated from amazon region. Our analysis suggest the presence of a plasmid similar to ChVi1 in two of these strains. The present work describes for the first time a biochemical characterization of RadA/Sms and RecA from C. violaceum which have different roles in HR. Moreover, the discovery of ChVi1 opens a path to further explore C. violaceum?s biology.

ATP plays a critical role in the regulation of many enzyme processes. In this work, I have focused on the ATP mediated regulation of the recombination processes catalyzed by the E. coliRecA and the human Rad51 proteins. The RecA protein is a multifunctional enzyme, which plays a central role in the processes of recombinational DNA repair, homologous genetic recombination and in the activation of the cellular SOS response to DNA damage. Each of these functions requires a common activating step, which is the formation of a RecA-ATP-ssDNA nucleoprotein filament. The binding of ATP results in the induction of a cooperative, high affinity ssDNA binding state within RecA (Menetski & Kowalczykowski, 1985b; Silver & Fersht, 1982). Data presented here identifies Gln194 as the NTP binding site "γ-phosphate sensor", in that mutations introduced at this residue disrupt all ATP induced RecA activities, while basal enzyme function is maintained. Additionally, we have dissected the parameters contributing to cooperative nucleoprotein filament assembly in the presence of cofactor. We show that the dramatic increase in the affinity of RecA for ssDNA in the presence of ATP is a result of a significant increase in the cooperative nature of filament assembly and not an increase in the intrinsic affinity of a RecA monomer for ssDNA. Previous work using both mutagenesis and engineered disulfides to study the subunit interface of the RecA protein has demonstrated the importance of Phe217 for the maintenance of both the structural and functional properties of the protein (Skiba & Knight, 1994; Logan et al., 1997; Skiba et al., 1999). A Phe217Tyr mutation results in a striking increase in cooperative filament assembly. In this work, we identify Phe217 as a key residue within the subunit interface and clearly show that Phe217 is required for the transmission of ATP mediated allosteric information throughout the RecA nucleoprotein filament. The human Rad51 (hRad51) protein, like its bacterial homolog RecA, catalyzes genetic recombination between homologous single and double stranded DNA substrates. This suggests that the overall process of homologous recombination may be conserved from bacteria to humans. Using IAsys biosensor technology, we examined the effect of ATP on the binding of hRad51 to ssDNA. Unlike RecA, we show that hRad51 binds cooperatively and with high affinity to ssDNA both in the presence and absence of nucleotide cofactor. These results show that ATP plays a fundamentally different role in hRad51 vs.RecA mediated processes. In summary, through the work presented in this dissertation, we have defined the critical molecular determinants for ATP mediated allosteric regulation within RecA. Furthermore, we have shown that ATP is not utilized by Rad51 in the same manner as shown for RecA, clearly defining a profound mechanistic difference between the two proteins. Future studies will define the requirement for ATP in hRad51 mediated processes.

Ce travail s'intéresse au rôle de l'hélicase UvrD dans le redémarrage des fourches de réplication inactivées chez Escherichia coli. Dans le cas d'arrêt de la réplication à un site Ter/Tus de terminaison de la réplication ectopique, nous avons montré que UvrD est l'hélicase majeure impliquée dans le redémarrage. Nous proposons que le rôle de l'hélicase UvrD lors du redémarrage des fourches bloquées à un site de terminaison ectopique consiste à déplacer le complexe Ter/Tus de terminaison lors du redémarrage de la réplication après recombinaison homologue. Nous avons pu montrer que ce rôle de UvrD est conservé chez son homologue PcrA de Bacillus subtilis. Dans le cas d'arrêt de la réplication après inactivation d'une sous-unité de la polymérase réplicative (Pol III) d'E. Coli, la sous-unité catalytique (mutant dnaEts) ou le facteur de processivité (mutant dnaNts), l'action anti-RecA de UvrD est essentielle à la réaction de réversion des fourches de réplication (RPR) permettant un redémarrage de la réplication. L'étude du mode d'action anti-RecA de UvrD aux fourches de réplication inactivées montre qu'il dépend de la sous-unité de Pol III inactivée. Dans le mutant dnaNts l'activité ATPase de UvrD est essentielle à son action anti-RecA. Au contraire, dans le mutant dnaEts l'action anti-RecA de UvrD ne nécessite pas l'activité ATPase de la protéine, et corrèle avec un besoin de la protéine RarA/MgsA pour la fixation de RecA. Par ailleurs, nous avons montré que l'action anti-RecA de UvrD aux fourches de réplication inactivées est conservé chez son homologue PcrA de Bacillus subtilis
This study aims at understanding the role of the UvrD helicase in the restart of arrested replication forks in Escherichia coli. After replication arrest at ectopic replication terminaison sites of Ter/Tus, we showed that UvrD is the major helicase needed to restart. We propose that UvrD acts in concert with homologous recombination proteins to dislodge Tus form Ter sites during replication restart. The Tus-removal action of UvrD is conserved in Bacillus subtilis homologous helicase PcrA. After replication fork arrest by the inactivation of a subunit of the DNA polymerase III holoenzyme (Pol Illh), either the catalytic subunit (dnaEts mutant) or the (3-clamp (dnaNts mutant), the anti-RecA action of UvrD at blocked forks is essential for the replication fork reversal reaction (RPR) to promote replication restart. We have shown that the anti-RecA action of UvrD at blocked forks reflects two different activities of this enzyme. An ATPase-deficient UvrD mutant is able to antagonize RecA in cells affected for the Pol IIIh catalytic subunit DnaE. In this mutant, RecA action at blocked forks specifically requires the protein RarA (MgsA). This suggests that UvrD acts by preventing RecA binding, possibly through counteracting RarA. In contrast, at forks affected for the Pol Illh clamp (DnaN), RarA is not required for RecA binding and the ATPase fonction of UvrD is essential to counteract RecA, supporting the idea that UvrD removes RecA from DNA. The anti-RecA action of UvrD at Pol IIIts-blocked forks is conserved in the Bacillus subtilis homologous helicase PcrA. Proliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches

Molte tecnologie sviluppate in campo diagnostico si basano sull’individuazione di specifici marcatori presenti nei campioni biologici. In questo ambito è ampiamente sfruttata la capacità intrinseca degli anticorpi di riconoscere il loro antigene. Pertanto, è fondamentale implementare i vari processi che permettono il rapido isolamento degli anticorpi e che ne garantiscono un alto livello di produzione. Grazie al continuo avanzamento delle tecnologie del DNA ricombinante, sono state sviluppate tecnologie in vitro che permettono l'isolamento su larga scala di anticorpi monoclonali. Il phage display è una tecnologia basata sullo screening di una libreria di anticorpi contro un dato antigene. La libreria di anticorpi è di solito composta da domini variabili delle catene leggere e pesanti di una immunoglobulina combinati casualmente, questi domini sono fusi con una proteina del capside del batteriofago M13. Ogni fago espone sulla sua superficie un singolo peptide, quindi si può facilmente risalire alla sequenza codificante dei frammenti anticorpali. L'obiettivo principale di questo lavoro è quello di ottimizzare l’intera pipeline che consente lo sviluppo di anticorpi monoclonali ricombinanti interessanti in ambito diagnostico. Questo processo inizia con l'isolamento di cloni in grado di riconoscere l'antigene partendo da una libreria fagica naïve. Pertanto, questo lavoro è iniziato con la creazione e la caratterizzazione di una libreria fagica naïve. Una volta costruita la libreria, questa è stata validata mediante NGS, in questo modo è stato possibile avere un'istantanea della sua diversità. La libreria è stata ulteriormente validata mediante il classico sequenziamento di Sanger in modo da poter anche valutare la qualità delle sequenze codificanti dei frammenti anticorpali che la compongono. Per ottimizzare e validare l'intera pipeline, come antigene “pilota” è stato scelto Interferone γ, una proteina rilevante da un punto di vista diagnostico perché è utilizzata nella diagnosi della tubercolosi. Diverse procedure di biopanning hanno permesso di isolare, complessivamente, 25 scFv diversi. Questi cloni sono stati testati anche su una piattaforma automatizzata progettata per i test immunodiagnostici e questo ne ha permesso una caratterizzazione più approfondita. In questo modo è stato possibile isolare i cloni che mostravano le prestazioni migliori. Tra questi, sono stati scelti due cloni per essere ulteriormente maturati in modo da migliorare la loro affinità nei confronti dell'antigene. Considerato che il ruolo chiave svolto dalla VH nel riconoscimento dell'antigene è ben noto, è stato deciso di creare due nuove librerie di display fagico composte da scFv tutti recanti la VH dei due cloni parentali e un pannello di VL. La procedura di selezione ha permesso di isolare due cloni che sembrano funzionare meglio rispetto il clone parentale quando testati sulla piattaforma automatizzata. In campo diagnostico, generalmente, sono utilizzate immunoglobuline nel loro formato nativo. Quindi, una volta selezionati i frammenti anticorpali, questi devono essere clonati in vettori che ne consentano l'espressione in linee cellulari di mammifero. In questo lavoro è stata costruita una serie di vettori che consentono l'espressione delle IgG, IgA, IgM umane e IgG di topo. Tutti i vettori sono stati trasfettati e la produzione di anticorpi è stata buona anche in termini di corretto folding dell'immunoglobulina. La fase finale di convalida dell'intera pipeline consiste nell'espressione dei tre cloni gli anti-interferone γ come immunoglobuline in formato nativo e nel test della loro funzionalità sulla piattaforma automatizzata come reagenti in un kit diagnostico della tubercolosi commerciale.
In diagnostics, many tools rely on the detection of specific markers in biological specimens and widely exploit the ability of antibodies to recognize their antigen. Thus, the implementation of the various processes ranging from antibody isolation to their high rate production is fundamental. Thanks to the advantages of recombinant DNA technologies, in vitro display methods have been developed for large scale isolation of monoclonal antibodies. Phage display is a technology based on the screening of an antibody library against a given antigen. The antibody library is usually composed of randomly combined immunoglobulin’s light and heavy variable domains fused with a coat protein of the bacteriophage M13. Each phage exposes on its surface a single peptide, so the coding sequence of the antibody fragments can be easily retrieved. The main objective of this work is to optimize a pipeline for recombinant monoclonal antibody development against antigens of diagnostic interest. This process begins with the isolation from a naïve phage library of clones able to recognize the antigen. Therefore, this work started with the creation and the characterization of a naïve phage display library. Once constructed the library, it has been validated with NGS to have an actual snapshot about library diversity and with Sanger sequencing to assess the quality of the coding sequences. To optimize and validate the whole pipeline, as a “pilot” antigen it has been chosen Interferon γ, a diagnostically relevant protein given its employment in the detection of tuberculosis. Different biopanning procedures allowed to isolate, overall, 25 different scFv. Thanks to a deeper characterization, performed on an automated platform designed for immunodiagnostic testing, it was possible to point out the best performing clones. Among them, two were chosen to be maturated to further enhance their affinity towards the antigen. Since it is well established the key role played by the VH in antigen recognition, it has been decided to create two new phage display libraries composed of scFv all bearing the VH of the two parental clones and a panel of VLs. The tailored selection procedure allowed to isolate two clones that seemed to perform better than the parental one on the automated platform. In a diagnostic setting, usually, full-size immunoglobulins are required, so, once selected, the antibody fragments are cloned in vectors allowing the expression in mammalian host cell lines. In this work a set of vectors allowing the expression of the human IgG, IgA, IgM and mouse IgG has been built. All the vectors have been transfected and antibody production was good also in terms of correct folding of the full-size immunoglobulin. The final step of validation of this whole pipeline consists in the expression of all the three anti-Interferon γ scFv as full-size immunoglobulin and the assay of their functionality on an automated platform as reagents in a commercial tuberculosis diagnostic kit.

Atualmente, o Brasil encontra-se na privilegiada posição de maior produtor e exportador mundial de carne bovina, tornando a pecuária uma das atividades nacionais mais importantes e rentáveis. Este dado enfatiza a importância de pesquisa e desenvolvimento em reprodução bovina, especialmente em hormônios estimuladores da ovulação, tais como a gonadotrofina coriônica equina (eCG). Os produtos comerciais à base de eCG comercialmente disponíveis são purificados a partir do sangue de éguas gestantes, apresentando variabilidade de lote para lote e presença de contaminantes. Estes fatos, juntamente com a limitação do material de partida (sangue equino), enfatizam a necessidade de haver um sistema de expressão de eCG recombinante passível de ser explorado comercialmente. Neste quesito, as células de mamíferos se mostram um sistema robusto para tal finalidade, visto que são capazes de adicionar modificações pós-traducionais às cadeias polipeptídicas, tais como a glicosilação, o que é essencial para o correto dobramento, maturação e montagem das duas subunidades, além de interferir diretamente com a meia-vida, o reconhecimento do receptor, a solubilidade e a atividade biológica das proteínas. No entanto, mesmo entre os sistemas de expressão heteróloga em células de mamífero, encontra-se muita variabilidade nos padrões de glicosilação adicionado. No presente trabalho, foi realizado um estudo comparativo através da clonagem e expressão de uma forma fusionada de eCG (reCGβα) em duas linhagens celulares diferentes: (1) CHO-DG44, um dos sistemas de expressão mais utilizados pelas indústrias farmacêuticas, capaz de adicionar N-glicanos complexos; e (2) 293T, uma linhagem humana capaz de produzir glicoproteínas carreando oligossacarídeos complexos e sialilados. Os resultados de atividade biológica (in vitro e in vivo) apontam uma maior atividade de reCG produzido por células CHO-DG44. O perfil de N-glicosilação de reCG produzido pelas células CHOD-G44 assemelhou-se mais à eCG selvagem, quando comparado a reCG produzido por células 293T. Por fim, estudos clínicos foram realizados com reCG produzido em meio livre de soro fetal bovino e parcialmente purificado, onde atividade específica de reCG produzido por células CHO-DG44 mostrou-se similar ao produto comercial selvagem.
Brazil is currently the major beef producer and exporter, rendering to livestock one of the country´s most economically relevant activities. This emphasizes the importance of research and development in bovine reproduction, especially at ovulation-stimulatory hormones, such as equine gonadotropin (eCG). The commercially available eCG-based products are purified from blood of pregnant heifers, presenting batch-to-batch variability and the presence of contaminants. These facts, together with the limitation of the bulk material (equine blood), emphasize the need of an eCG expression system able to be commercially explored. In this aspect, mammalian cells are a robust system, capable of add post-translational modifications to polypeptide chains, such as glycosylation, which is essential for the correct folding, maturation and assembly of both eCG subunits. In addition, glycosylation directly interferes with the protein half-life, receptor recognition, solubility and biological activity. In the present work, a comparative study was carried out by cloning and expressing a fusion form of eCG (reCGβα) in two different mammalian cell lines: (1) CHO-DG44, one of the most used by pharmaceutical companies expression systems, capable of add complex-type N-glycans; and (2) 293T, a human cell line capable of produce glycoproteins carrying complex and sialylated oligosaccharides. The in vitro and in vivo biological activity results show a higher potency of reCG produced by CHO-DG44 cells. The N-glycosylation pattern produced by CHO-DG44 cells was more similar to native eCG in comparison to the N-glycosylation produced by 293T cells. Finally, clinical studies were performed with serum absent media produced and partially purified reCG, showing that the specific activity of reCG produced by CHO cells was similar to the commercial wild type product.

Maintenir l'intégrité du matériel génétique de toute cellule vivante face à des altérations d'origine endogène ou exogène, est un problème crucial auquel tout organisme est confronté. Les lésions induites dans l'ADN peuvent interférer avec des processus tels que la réplication et la transcription et entraîner une désorganisation de l'activité cellulaire pouvant conduire à la mort de la cellule. J'ai caractérisé l'expression du gène humain KIN17 après traitement par différents agents génotoxiques. La protéine KIN17 possède une région centrale homologue à un domaine de fixation à l'ADN dans la partie C-terminale de la protéine RecA d'E. Coli. RecA est essentielle à la recombinaison génétique, à la réponse cellulaire aux rayonnements et à la mutagenèse. Mes résultats indiquent une participation active de la protéine KIN17 dans la réponse aux dommages de l'ADN produit par les ultraviolets C (UVC) et les rayonnements gamma (γ). Les cinétiques d'expression du gène KIN17 sont différentes selon la nature de l'agent génotoxique. Compte tenude mes résultats, j'ai cherché à identifier les mécanismes responsables de cette réponse au stress génotoxique en utilisant des lignées cellulaires mutées pour le gène p53 et des cellules exprimant un mutant dominant négatif du facteur de transcription ATF2 : J'ai constaté que l'augmentation de l'expression du gène KIN17 était indépendante de p53 après irradiation γ et UVC. En revanche, ATF2 semble contrôler l’expression du gène KIN17 après γ. L'analyse de l'expression du gène KIN17 dans des cellules déficientes dans la réparation par excision de l'ADN, indique qu'une réparation efficace est indispensable à l'augmentation transitoire de l'expression du gène KIN17 après irradiation aux UVC. Toutes ces observations montrent que le gène KIN17 intervient dans une voie de signalisation qui pourrait aider à contrebalancer les effets délétères des agents génotoxiques. Des résultats préliminaires sur des hépatocarcinomes humains montrent une augmentation de l'expression du gène KIN17 lors de la progression tumorale
All organisms are confronted by the crucial problem of protecting the integrity of the genetic material in their cells against alterations provoked by endogenous or exogenous agents. DNA damage may interfere with essential processes such as replication and transcription, thus leading to metabolic disruption or to cell death. Ihave characterized the expression profile of KIN17 gene after treatment with different genotoxic agents. KIN17 protein possesses a core region homologous to the DNA-binding domain located in the C-terminal part of the E. Coli RecA protein. RecA plays an essential role in the cellular response to radiation, in recombination and in mutagenesis. My results indicate that the human kin17 protein actively participates in the cellular response to the DNA damage produced by UVC- and γ-irradiation. The kinetics of KIN17 gene expression differs according to the nature of the genotoxic agent. Considering these results, I tried to identify the mechanisms responsible for this response to genotoxic stress by using cells mutated in the p53 gene or cells expressing a dominant negative mutant for ATF2. I noticed that the increase in KIN17 gene expression was independent of p53. The transcription factor ATF2, on the other hand, appeared to be involved in the control of KIN17 gene expression after γ-irradiation. Using cells deficient for nucleotide excision repair (NER), I have demonstrated that an active NER is necessary for the transient increase in KIN17 gene expression after UVC-irradiation. Taken together, these data indicate the Participation of KIN17 gene in a signalling pathway that may help to counterbalance the deleterious effects of genotoxic agents. Prelirninary results on human hepatocarcinoma show increased expression levels of KIN17 gene during tumoral progression

Homologous genetic recombination, because of its fundamental roles in the maintenance of genome stability and evolution, is an essential cellular function common to all organisms. This process also plays important roles in the repair of damaged DNA molecules, generation of genetic diversity and proper segregation of chromosomes. The genetic exchange is a highly orchestrated process that entails a plethora of control mechanisms and a large number of proteins, of which RecA and SSB are two proteins that have been chosen for further investigation(s) in the present study. In addition, we have also investigated the interaction between SSB and UvrD1, which plays an important role in DNA repair pathways, especially nucleotide excision repair (NER) and mismatch repair as well as DNA replication and recombination. Chapter 1 reviews the literature regarding various aspects of homologous recombination, with an emphasis on the biochemical and the biophysical aspects of RecA and SSB proteins. In addition, it provides an overview of the study of DNA repair and recombination in mycobacteria. RecA protein is ubiquitous and well conserved among bacterial species. Many archaeal species possess two RecA homologues (RadA and RadB) and eukarya possess multiple homologues of RecA including, Rad51, Rad51B, Rad51C, Rad51D, DMC1, XRCC2, or XRCC3. RecA or its homologues function as polymers, consisting of hundreds of monomers that cooperatively polymerize on single-stranded DNA to form a nucleoprotein filament. E. coli RecA protein participates in Trans Lesion Synthesis (TLS) of DNA and forms the minimal mutasome in association with DNA polymerase V (UmuD’2C). The fundamental mechanism underlying HR, i.e. DNA strand exchange, is one of the most fascinating examples of molecular recognition and exchange between biological macromolecules. Since the isolation of E. coli recA gene and the subsequent purification of its gene product and also from other organisms, RecA protein has been studied extensively for more than three decades. E. coli RecA protein has pivotal roles in DNA recombination and repair, and binding to DNA in the presence of ATP, is a fundamental property of RecA protein resulting in the formation of a nucleoprotein filament. This is the slow step of the HR process, and is considerably faster on ssDNA than on duplex DNA. Binding of RecA to dsDNA is slower at physiological pH, is accelerated at acidic pH, and the lag in binding at the higher pH values is due to slow nucleation. The ATP and the DNA binding functions of RecA display allosteric interaction such that ATP- binding leads to an increase in affinity to ssDNA-binding and vice-versa. X-ray structures of E. coli RecA complexed with nucleotide cofactors have implicated a highly conserved Gln196 in Mycobacterium smegmatis RecA in the coupling of ATP and the DNA binding domains. The carboxyamide group of Gln196 makes an H-bond with the γ-phosphate group of ATP and the side chain of this residue is observed to move by approximately 2Å towards the ATP, relative to the other residues involved in ATP binding. In addition, a highly conserved Arg198 has also been postulated to interact with the γ-phosphate group of bound ATP and position it for a nucleophilic attack by a conserved residue-Glu96 leading to ATP hydrolyses. To elucidate the role of Gln196 and Arg198 in the allosteric modulation of RecA functions, we generated MsRecA variant proteins, where in Gln196 was substituted with alanine, asparagine or glutamate; Arg198 was mutated to a lysine. The biochemical characterization of MsRecA and its variant proteins with the objective of defining the allosteric interaction between the ATP- and the DNA-binding sites has been described with in Chapter 2. We observed that while the mutant MsRecA proteins were proficient in ATP-binding they were deficient in ATP hydrolyses. We assayed for the ability of these proteins to bind ssDNA using either nitrocellulose filter binding or Surface Plasmon Resonance (SPR). While we did not detect any ssDNA-binding by the mutant MsRecA proteins in the filter binding assay, we observed only ten-fold reduction in the affinity for ssDNA as compared to wild type MsRecA protein in MsRecAQ196A, Q196N and R198K in the SPR assay. MsRecA Q196E did not show any binding to ssDNA, in both nitrocellulose filter-binding as well as SPR assays. We assayed for the ability of the mutant RecA proteins for their ability to promote DNA-pairing as well as DNA strand exchange. While we observed limited pairing promoted by the mutant proteins relative to the wild-type MsRecA, we observed a complete abrogation of strand exchange in the case of mutant proteins. In addition, we assayed for the co-protease function of MsRecA, by monitoring the cleavage of MtLexA. We observed that only the wild-type MsRecA protein was able to cleave MtLexA, while none of the mutant RecA proteins were able to do so. In order to understand the differences observed between the wild -type and the mutant MsRecA proteins, we analyzed the conformational state of MsRecA and its variant proteins by circular dichroism spectroscopy upon ATP-binding. We observed that while MsRecA and MsRecAQ196N displayed a reduction in the absorbance at 220 nm upon ATP binding, we did not observe any such structural transitions in the other mutant MsRecA proteins that we tested. Based on our observations and the crystal structure of E. coli RecA bound to ssDNA, in Chapter 2, we propose a dual role for the Gln196 and Arg198 in modulating RecA activities. In the presynaptic filament Gln196 and Arg198 sense the presence of the nucleotide in the nucleotide binding pocket and initiate a series of conformation changes that culminate in the transition to an active RecA nucleoprotein filament. In the active RecA nucleoprotein filament these residues are repositioned such that they now form a part of the protomer-protomer interface. As such they perform two vital functions; they stabilize the protomer-protomer interface by participating in the formation of hydrogen bonds that span the interface as well transmit the wave of ATP hydrolysis across the interface leading to a coordinated hydrolyses of ATP essential for the heteroduplex extension phase of strand exchange reaction. The members of the super family of single stranded DNA binding proteins (SSB) play an important role in all aspects of DNA metabolism including DNA replication, repair, transcription and recombination. Prokaryotic SSBs bind ssDNA with high affinity and generally with positive cooperativity. Several lines of evidence suggest that prokaryotic SSBs are modularly organized into three distinct domains: the N-terminal DNA binding domain and acidic C-terminal domain are linked by a flexible spacer. Studies from our laboratory have revealed that M. smegmatis SSB plays a concerted role in recombination-like activities promoted by the cognate RecA. The C- terminal of SSB is known to be involved in its ability to interact with other proteins. We have previously reported that the C-terminal domain of M. smegmatis SSB, which is not essential for interaction with DNA, is the site for the binding of cognate RecA. The data in Chapter 3 describes the characterization of the SSB C-terminus with the objective of delineating the elements responsible for mediating protein-protein interaction, as well as to define the mechanism by which SSB is able to modulate the activities of RecA. To map the RecA interaction domain of SSB we created deletion mutants in MsSSB lacking 5, 10, 15 or 20 residues from the C-terminal. The truncated SSB proteins were expressed with a His- tag at the N- terminus and purified to homogeneity using a Ni-NTA affinity matrix. We observed unlike MsSSB, MsSSB∆C5 and MsSSB∆C10, MsSSB∆C15 and MsSSB∆C20 were unable to support three-strand exchange catalyzed by MsRecA. Based on the observation that interaction with SSB is essential for MsRecA to catalyze the strand Exchange reaction, we postulate that the RecA interacting domain of SSB is situated between the 15th and the 20th residue from the C-terminal. Further, the C-terminal of MsSSB modulates the transitions between DNA binding modes. Unlike the case with EcSSB where deletion of the last 8 residues from the C-terminal stabilizes the (SSB)35 mode of ssDNA binding, we observe that in case of MsSSB the deletion of C-terminal seems to destabilize the (SSB)35. In addition, the transition from the low density binding mode to a high density mode involves the formation of several intermediates when the C-terminal residues are deleted. With the objective of understanding the functions to the C-terminal of SSB independent of its DNA-binding domain in modulating RecA functions, we employed a peptide corresponding to the 35 residues from the C-terminal of the MsSSB. We observed that the C-terminal region alone is capable of interacting with RecA. In addition we also observed that the C-terminal domain of SSB stimulates RecA functions independent of its DNA binding domain. To address the question, whether the stimulatory effect of the C-terminal domain of SSB in the absence of its DNA-binding domain is restricted to RecA or is a generalized phenomenon associated with all SSB interacting proteins; we tested the effect of C-terminal domain of SSB on UvrD which is known to interact with SSB. UvrD participates in several pathways of DNA metabolism, which include the nucleotide excision repair (NER) and mismatch repair pathway, replication and recombination. Genetic evidence suggests that UvrD and SSB interact in vivo. We tested the effect of mycobacterial SSB on M. tuberculosis UvrD1 (MtUvrD1) functions in vitro. We observe that MtUvrd1 physically interacts with SSB. Further, presence of SSB has an inhibitory effect on the helicase activity of MtUvrD1 and that this effect is dependent on the C-terminal region as the deletion of residues from the C-terminal of SSB abrogates the inhibitory effect of SSB. However, unlike RecA, the C-terminal region of SSB alone had no effect on the helicase activity of UvrD1. We also observed that MsSSB has opposing effects on the ATPase activity of MtUvrD1. In the presence of low concentrations of SSB the ATPase activity is enhanced, while we observed an inhibition when the concentration of MsSSB is high. The precise mechanistic details of how SSB is able to act as an accessory protein to RecA, in context of homologous recombination and stimulates its biochemical activities have been a subject of debate. Whereas research from some groups has shown that the stimulatory effect SSB is mediated through its ability to melt DNA secondary structure, thereby allowing RecA to overcome the kinetic barrier imposed by the presence of secondary structure in ssDNA, others postulate that SSB plays a direct role in the stabilization of RecA nucleoprotein filament and prevents its dissociation. Chapter 3 discusses the experimental evidence in favor of the aforesaid models and based on the results of our experiments; we propose that the accessory functions of SSB may be mediated by a mechanism that involves elements of both models. While interaction with SSB can bring about a conformational change in RecA that is reflected in the enhanced levels of strand exchange and co-protease activity, the helix destabilizing function of SSB is essential during heteroduplex extension and to sequester the displaced strand such that it does not participate in any further pairing reactions. The novel finding that we present in Chapter 3 is that the interaction of SSB C-terminal alone has a stimulatory effect upon RecA activities. Furthermore, we observed that M. tuberculosis UvrD1 is a weak interaction partner of SSB. The physical and functional interactions between MsSSB with RecA on the one hand, and MsSSB and UvrD1 on the other highlight different types of cross-talk between the components of HR and DNA repair pathways. In contrast to the results of earlier studies, our results indicate that protein-protein interactions alone between SSB and RecA may modulate the RecA mediated processes of presynapsis, homologous pairing and strand exchange between homologous DNA molecules as well as modulate its co-protease activity. In addition, our studies indicate that a direct protein-protein interaction is responsible for the modulation of UvrD1 activities by SSB.

Homologous genetic recombination, because of its fundamental roles in the maintenance of genome stability and evolution, is an essential cellular function common to all organisms. This process also plays important roles in the repair of damaged DNA molecules, generation of genetic diversity and proper segregation of chromosomes. The genetic exchange is a highly orchestrated process that entails a plethora of control mechanisms and a large number of proteins, of which RecA and SSB are two proteins that have been chosen for further investigation(s) in the present study. In addition, we have also investigated the interaction between SSB and UvrD1, which plays an important role in DNA repair pathways, especially nucleotide excision repair (NER) and mismatch repair as well as DNA replication and recombination. Chapter 1 reviews the literature regarding various aspects of homologous recombination, with an emphasis on the biochemical and the biophysical aspects of RecA and SSB proteins. In addition, it provides an overview of the study of DNA repair and recombination in mycobacteria. RecA protein is ubiquitous and well conserved among bacterial species. Many archaeal species possess two RecA homologues (RadA and RadB) and eukarya possess multiple homologues of RecA including, Rad51, Rad51B, Rad51C, Rad51D, DMC1, XRCC2, or XRCC3. RecA or its homologues function as polymers, consisting of hundreds of monomers that cooperatively polymerize on single-stranded DNA to form a nucleoprotein filament. E. coli RecA protein participates in Trans Lesion Synthesis (TLS) of DNA and forms the minimal mutasome in association with DNA polymerase V (UmuD’2C). The fundamental mechanism underlying HR, i.e. DNA strand exchange, is one of the most fascinating examples of molecular recognition and exchange between biological macromolecules. Since the isolation of E. coli recA gene and the subsequent purification of its gene product and also from other organisms, RecA protein has been studied extensively for more than three decades. E. coli RecA protein has pivotal roles in DNA recombination and repair, and binding to DNA in the presence of ATP, is a fundamental property of RecA protein resulting in the formation of a nucleoprotein filament. This is the slow step of the HR process, and is considerably faster on ssDNA than on duplex DNA. Binding of RecA to dsDNA is slower at physiological pH, is accelerated at acidic pH, and the lag in binding at the higher pH values is due to slow nucleation. The ATP and the DNA binding functions of RecA display allosteric interaction such that ATP- binding leads to an increase in affinity to ssDNA-binding and vice-versa. X-ray structures of E. coli RecA complexed with nucleotide cofactors have implicated a highly conserved Gln196 in Mycobacterium smegmatis RecA in the coupling of ATP and the DNA binding domains. The carboxyamide group of Gln196 makes an H-bond with the γ-phosphate group of ATP and the side chain of this residue is observed to move by approximately 2Å towards the ATP, relative to the other residues involved in ATP binding. In addition, a highly conserved Arg198 has also been postulated to interact with the γ-phosphate group of bound ATP and position it for a nucleophilic attack by a conserved residue-Glu96 leading to ATP hydrolyses. To elucidate the role of Gln196 and Arg198 in the allosteric modulation of RecA functions, we generated MsRecA variant proteins, where in Gln196 was substituted with alanine, asparagine or glutamate; Arg198 was mutated to a lysine. The biochemical characterization of MsRecA and its variant proteins with the objective of defining the allosteric interaction between the ATP- and the DNA-binding sites has been described with in Chapter 2. We observed that while the mutant MsRecA proteins were proficient in ATP-binding they were deficient in ATP hydrolyses. We assayed for the ability of these proteins to bind ssDNA using either nitrocellulose filter binding or Surface Plasmon Resonance (SPR). While we did not detect any ssDNA-binding by the mutant MsRecA proteins in the filter binding assay, we observed only ten-fold reduction in the affinity for ssDNA as compared to wild type MsRecA protein in MsRecAQ196A, Q196N and R198K in the SPR assay. MsRecA Q196E did not show any binding to ssDNA, in both nitrocellulose filter-binding as well as SPR assays. We assayed for the ability of the mutant RecA proteins for their ability to promote DNA-pairing as well as DNA strand exchange. While we observed limited pairing promoted by the mutant proteins relative to the wild-type MsRecA, we observed a complete abrogation of strand exchange in the case of mutant proteins. In addition, we assayed for the co-protease function of MsRecA, by monitoring the cleavage of MtLexA. We observed that only the wild-type MsRecA protein was able to cleave MtLexA, while none of the mutant RecA proteins were able to do so. In order to understand the differences observed between the wild -type and the mutant MsRecA proteins, we analyzed the conformational state of MsRecA and its variant proteins by circular dichroism spectroscopy upon ATP-binding. We observed that while MsRecA and MsRecAQ196N displayed a reduction in the absorbance at 220 nm upon ATP binding, we did not observe any such structural transitions in the other mutant MsRecA proteins that we tested. Based on our observations and the crystal structure of E. coli RecA bound to ssDNA, in Chapter 2, we propose a dual role for the Gln196 and Arg198 in modulating RecA activities. In the presynaptic filament Gln196 and Arg198 sense the presence of the nucleotide in the nucleotide binding pocket and initiate a series of conformation changes that culminate in the transition to an active RecA nucleoprotein filament. In the active RecA nucleoprotein filament these residues are repositioned such that they now form a part of the protomer-protomer interface. As such they perform two vital functions; they stabilize the protomer-protomer interface by participating in the formation of hydrogen bonds that span the interface as well transmit the wave of ATP hydrolysis across the interface leading to a coordinated hydrolyses of ATP essential for the heteroduplex extension phase of strand exchange reaction. The members of the super family of single stranded DNA binding proteins (SSB) play an important role in all aspects of DNA metabolism including DNA replication, repair, transcription and recombination. Prokaryotic SSBs bind ssDNA with high affinity and generally with positive cooperativity. Several lines of evidence suggest that prokaryotic SSBs are modularly organized into three distinct domains: the N-terminal DNA binding domain and acidic C-terminal domain are linked by a flexible spacer. Studies from our laboratory have revealed that M. smegmatis SSB plays a concerted role in recombination-like activities promoted by the cognate RecA. The C- terminal of SSB is known to be involved in its ability to interact with other proteins. We have previously reported that the C-terminal domain of M. smegmatis SSB, which is not essential for interaction with DNA, is the site for the binding of cognate RecA. The data in Chapter 3 describes the characterization of the SSB C-terminus with the objective of delineating the elements responsible for mediating protein-protein interaction, as well as to define the mechanism by which SSB is able to modulate the activities of RecA. To map the RecA interaction domain of SSB we created deletion mutants in MsSSB lacking 5, 10, 15 or 20 residues from the C-terminal. The truncated SSB proteins were expressed with a His- tag at the N- terminus and purified to homogeneity using a Ni-NTA affinity matrix. We observed unlike MsSSB, MsSSB∆C5 and MsSSB∆C10, MsSSB∆C15 and MsSSB∆C20 were unable to support three-strand exchange catalyzed by MsRecA. Based on the observation that interaction with SSB is essential for MsRecA to catalyze the strand Exchange reaction, we postulate that the RecA interacting domain of SSB is situated between the 15th and the 20th residue from the C-terminal. Further, the C-terminal of MsSSB modulates the transitions between DNA binding modes. Unlike the case with EcSSB where deletion of the last 8 residues from the C-terminal stabilizes the (SSB)35 mode of ssDNA binding, we observe that in case of MsSSB the deletion of C-terminal seems to destabilize the (SSB)35. In addition, the transition from the low density binding mode to a high density mode involves the formation of several intermediates when the C-terminal residues are deleted. With the objective of understanding the functions to the C-terminal of SSB independent of its DNA-binding domain in modulating RecA functions, we employed a peptide corresponding to the 35 residues from the C-terminal of the MsSSB. We observed that the C-terminal region alone is capable of interacting with RecA. In addition we also observed that the C-terminal domain of SSB stimulates RecA functions independent of its DNA binding domain. To address the question, whether the stimulatory effect of the C-terminal domain of SSB in the absence of its DNA-binding domain is restricted to RecA or is a generalized phenomenon associated with all SSB interacting proteins; we tested the effect of C-terminal domain of SSB on UvrD which is known to interact with SSB. UvrD participates in several pathways of DNA metabolism, which include the nucleotide excision repair (NER) and mismatch repair pathway, replication and recombination. Genetic evidence suggests that UvrD and SSB interact in vivo. We tested the effect of mycobacterial SSB on M. tuberculosis UvrD1 (MtUvrD1) functions in vitro. We observe that MtUvrd1 physically interacts with SSB. Further, presence of SSB has an inhibitory effect on the helicase activity of MtUvrD1 and that this effect is dependent on the C-terminal region as the deletion of residues from the C-terminal of SSB abrogates the inhibitory effect of SSB. However, unlike RecA, the C-terminal region of SSB alone had no effect on the helicase activity of UvrD1. We also observed that MsSSB has opposing effects on the ATPase activity of MtUvrD1. In the presence of low concentrations of SSB the ATPase activity is enhanced, while we observed an inhibition when the concentration of MsSSB is high. The precise mechanistic details of how SSB is able to act as an accessory protein to RecA, in context of homologous recombination and stimulates its biochemical activities have been a subject of debate. Whereas research from some groups has shown that the stimulatory effect SSB is mediated through its ability to melt DNA secondary structure, thereby allowing RecA to overcome the kinetic barrier imposed by the presence of secondary structure in ssDNA, others postulate that SSB plays a direct role in the stabilization of RecA nucleoprotein filament and prevents its dissociation. Chapter 3 discusses the experimental evidence in favor of the aforesaid models and based on the results of our experiments; we propose that the accessory functions of SSB may be mediated by a mechanism that involves elements of both models. While interaction with SSB can bring about a conformational change in RecA that is reflected in the enhanced levels of strand exchange and co-protease activity, the helix destabilizing function of SSB is essential during heteroduplex extension and to sequester the displaced strand such that it does not participate in any further pairing reactions. The novel finding that we present in Chapter 3 is that the interaction of SSB C-terminal alone has a stimulatory effect upon RecA activities. Furthermore, we observed that M. tuberculosis UvrD1 is a weak interaction partner of SSB. The physical and functional interactions between MsSSB with RecA on the one hand, and MsSSB and UvrD1 on the other highlight different types of cross-talk between the components of HR and DNA repair pathways. In contrast to the results of earlier studies, our results indicate that protein-protein interactions alone between SSB and RecA may modulate the RecA mediated processes of presynapsis, homologous pairing and strand exchange between homologous DNA molecules as well as modulate its co-protease activity. In addition, our studies indicate that a direct protein-protein interaction is responsible for the modulation of UvrD1 activities by SSB.

La stabilité génomique des organelles de plantes suscite un grand intérêt dans le domaine de la biologie végétale. En effet, plusieurs études récentes suggèrent que ce type d’instabilité génomique pourrait mener à l’isolation de traits intéressants en l’agronomie. Plusieurs protéines sont d’ailleurs déjà été identifiés comme étant impliqués dans le maintien de la stabilité de ces génomes, tels que MSH1, la famille des POLI, OSB1, les protéines Whirly et les Recombinases A (RECA). Le génome nucléaire d’Arabidopsis thaliana encode trois protéines s’apparentant à la Recombinase A bactérienne et qui sont ciblées à la mitochondrie et/ou au chloroplaste, soit RECA1, RECA2 et RECA3. Globalement, ces gènes partagent une similarité de séquence de 61% avec leur homologue bactérien chez Escherichia coli. Chez les bactéries ces protéines jouent un rôle essentiel dans la recombinaison homologue et sont impliquées dans la réparation de l’ADN. Chez Arabidopsis, il a été démontré que RECA2 et RECA3 sont nécessaires au maintien de l’intégrité du génome mitochondriale. Toutefois leur contribution à la stabilité du génome chloroplastique ainsi que le rôle de RECA1 restent obscures. Le but de ce projet est donc de déterminer la contribution éventuelle des protéines RECA d’Arabidopsis dans la réparation de l’ADN chloroplastique et plus précisément le rôle du gène RECA1. Nous énonçons l’hypothèse que les RECA de plantes se comportent effectivement comme leurs orthologues bactériens en étant impliqués dans la recombinaison homologue. Dans le cadre de ce projet, nous avons tenté d’isoler des lignées mutantes pour chacun des gènes RECA d’Arabidopsis. En somme, nous avons pu obtenir des lignées convenables pour notre étude que dans le cas du gène RECA1. Ces lignées ont été utilisées pour évaluer la contribution de ce gène à la stabilité du génome du chloroplaste. Ensuite, pour étudier la relation épistatique des gènes RECA1, WHY1 et WHY3, un croisement des différentes lignées mutantes pour ces gènes a été réalisé. Nous avons ensuite étudié la sensibilité de toutes ces lignées mutantes à la ciprofloxacine, un agent causant des bris double brin exclusivement dans les organelles de plantes. Finalement, iii nous avons testé la présence de réarrangements dans le génome du chloroplaste en condition normal ou en présence de stress génotoxique. Nos résultats démontrent que les protéines Whirly et RECA1 sont impliquées dans deux voies de réparation de l’ADN différentes et que les Whirly sont suffisantes pour s’occuper des bris d’ADN double brin en l’absence de RECA1. Nous démontrons également que l’absence de Whirly et RECA1 entraine une forte augmentation de la quantité de réarrangements dans le génome du chloroplaste. De plus nous proposons que la polymérase POLIB est impliquée dans la même voie de réparation que RECA1. Finalement nous proposons un modèle pour expliquer nos résultats et impliquons RECA1 dans un mécanisme de réparation d’ADN et aussi un rôle potentiel dans la réplication.
The stability of plant organelles genomes elicits a great interest in the domain of plant biology. In fact, numerous studies suggest that genomic instability can lead to the isolation of interesting traits in the field of agronomy. Some factors such as MSH1, the POLI family, OSB1, the Whirly proteins and the Recombinase A (RECA), have already been identified has being implicated in the maintenance of genome stability. The nuclear genome of Arabidopsis thaliana encodes three proteins, RECA1, RECA2 and RECA3, that shares a high resemblance with bacterial Recombinase A. They are targeted to the mitochondria and/or to the chloroplast. Globally, these genes share a similarity of sequence of 61% with their bacterial homologue in Escherichia coli. In bacteria these proteins play an essential part in homologous recombination and are implicated in DNA repair. In Arabidopsis, RECA2 and RECA3 have been shown as being essential to maintain the integrity of the mitochondrial genome but their contribution to the stability of the chloroplast as well as the role of RECA1 remains obscure. The goal of this project is to establish the eventual contribution of the Arabidopsis RECA proteins in the repair of chloroplast DNA and more precisely the role of the RECA1 gene. We propose the hypothesis that plants RECA act in the same fashion as their bacterial orthologues by being implicated in homologous recombination. Within the framework of this project, we have attempted to isolate mutant lines for each RECA gene of Arabidopsis. In the end, we were able to obtain appropriate lines for our study only for the RECA1 gene. These lines were then used to evaluate the contribution of the gene to chloroplast genome stability. Afterwards, in order to study the epistatic relationship between the RECA1, WHY1 and WHY3 genes, a cross between different mutant lines of these genes was realised. We then studied the sensitivity of all of those mutant lines to ciprofloxacine, an agent causing double stranded breaks exclusively in plant organelles. Finally, we evaluated the presence of rearrangements in the chloroplast genome under normal conditions and under the presence of a genotoxic stress. Our v results show that the Whirly and RECA1 proteins are implicated in two separate pathways of DNA reparation and that the Whirly proteins are sufficient to take in charge DNA double strand breaks generated by the absence of RECA1. We also demonstrate that the absence of Whirly and RECA1 causes an increasein the quantity of rearrangements in the chloroplast genome. Furthermore, we propose that the polymerase POLIB is implicated in the same repair pathway as RECA1. Finally we propose a model to explain our results and implicate RECA1 in a DNA repair mechanism and propose a role for RECA1 in DNA replication.

博士
臺灣大學
生化科學研究所
98
This thesis focused on two subjects. One is the study on structure and function of Sulfolobus solfataricus (Sso) RadA. Another is the peptide (IRFLTARRR) derived from structure of RecA-DNA complex can promote not only the enzymatic activity of RecA protein but also resistance to DNA damaging agents. The RecA family of proteins is essential in homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. In the previous reports, RecA family of proteins is thought to perform DNA strand exchange as a right-handed filament (active form) or as a closed-ring (inactive form). In this thesis, we report two new crystal structures that are left-handed and overwound right-handed helical filaments. Comparing the four different structures, we suppose that the DNA homology pairing and strand exchange occurs in the overwound right-handed nucleoprotein filament, and release of DNA exchange final products using the left-handed filament. We also identified the conserved hinge region (subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. The results of several in vitro experiments are consistent with our hypothesis. Another story is about a rationally-designed small peptide based on the Escherichia coli RecA-DNA crystal structure can promote homologous recombination through the enhancement of both RecA-mediated strand assimilation and three-strand exchange activity. We identified that the hydrophobicity and poly-positive charges, and the space between them in those small peptides are crucial features for such activities. Remarkably, peptide #3 alone without RecA can also promote the D-loop formation at elevated temperature. Cell viability assays showed that the peptide elevates mammalian cell resistance to two cytotoxic DNA drugs, cisplatin and doxorubicin. The rescue of viability may result from increased DNA repair efficiency. Such peptides may find future biological applications.