Dissertations / Theses on the topic 'RecG'

Efficient and accurate repair of DNA double strand breaks (DSBs) is required to maintain genomic stability in both eukaryotes and prokaryotes. In Escherichia coli, DSBs are repaired by homologous recombination (HR). During this process, DNA synthesis needs to be primed and templated from an intact homologous sequence to restore any information that may have been lost on the broken DNA molecule. Two critical late stages of the pathway are repair DNA synthesis and the processing of Holliday junctions (HJs). However, our knowledge of the detailed mechanisms of these steps is still limited. Our laboratory has developed a system that permits the induction of a site-specific DSB in the bacterial chromosome. This break forms in a replication dependent manner on one of the sister chromosomes, leaving the second sister chromosome intact for repair by HR. Unlike previously available systems, the repairable nature of these breaks has made it possible to physically investigate the different stages of DNA double-strand break repair (DSBR) in a chromosomal context. In this thesis, I have addressed some fundamental questions relating to repair DNA synthesis and processing of HJs by using a combination of mutants defective in specific biochemical reactions and an assay that I have developed to detect repair DNA synthesis, using a polar termination sequence (terB). First, by using terB sites located at different locations around the break point, it was shown that the DnaB-dependent repair forks are established in a coordinated manner, meaning that the collision of the repair forks occurs between two repair DNA synthesis initiation sites. Second, DSBR was shown to require the PriB protein known to transduce the DNA synthesis initiation signal from PriA protein to DnaT. Conversely, the PriC protein (known as an alternative to PriB in some reactions) was not required in this process. PriB was also shown to be required to establish DnaB-dependent repair synthesis using the terB assay. Third, the establishment and termination of repair DNA synthesis by collision of converging repair forks were shown to occur independently of HJ resolution. This conclusion results from the comparison of the viability of single and double mutants, deficient in either the establishment of DNA synthesis, HJ resolution or in both reactions, subjected to DSBs and from the study of the DNA intermediates that accumulated in these mutants as detected by two-dimensional gel electrophoresis. Fourth, the role of RecG protein during DSB repair was investigated. Solexa sequencing analyses showed that recG null mutant cells undergoing DSBs accumulate more DNA around the break point (Mawer and Leach, unpublished data). This phenomenon was further investigated by two different approaches. Using terB sites in different locations around the break point and ChIP-Seq analyses to investigate the distribution of RecA in a recG null mutant demonstrating that the establishment of repair forks depends on the presence of RecG. Further studies using PriA helicase-dead mutant showed that the interplay between RecG and PriA proteins is essential for the establishment of correctly oriented repair forks during DSBR. As a whole, this work provides evidence on the coordinated nature of the establishment and termination of DNA synthesis during DSBR and how this requires a correct interplay between PriA-PriB and RecG. A new adapted model of homologous recombination is presented.

Ce travail présente l'étude à l'echelle de la molécule unique la jonction de Holliday, et de ses interactions avec les protéines MutS et ReG. Nous commençons par le comportement de la jonction de Holliday. La présence au niveau du centre de la jonction d'une hétérologie entre les séquences suffit à bloquer la migration sous certaines conditions expérimentales. Ce phénomène nous permet de détecter les hétérologies de séquences entre deux molécules. Nous avons étudié l'interaction entre une molécule d'ADN présentant une jonction de Holliday, et la protéine MutS. La présence de MutS bloque la migration lorsqu’un mésappariement arrive au niveau du centre de la jonction. La protéine RecG est une hélicase qui intervient dans le sauvetage des fourches de réplication. Des études suggèrent sa capacité à faire rebrousser les fourches de réplication arrêtées. Nous avons étudié ces réactions à l'échelle de la molécule unique. Nous avons observé la migration de la jonction. Cela nous a permis d'effectuer une mesure directe de la vitesse de l'enzyme, sa processivité et le couple maximal fourni par l'enzyme. Nous avons étudié la dépendance en concentration d'ATP et d'enzyme

Genomic instability driven by non-allelic homologous recombination (NAHR) provides a realistic mechanism that could account for the numerous chromosomal abnormalities that are hallmarks of cancer. We recently demonstrated that this type of instability could be assayed by analyzing the copy number variation of the human ribosomal RNA gene clusters (rDNA). Further, we found that gene cluster instability (GCI) was present in greater than 50% of the human cancer samples that were tested. Here, data is presented that confirms this phenomenon in the human GAGE gene cluster of those cancer patients. This adds credence to the hypothesis that NAHR could be a driving force for carcinogenesis. This data is followed by experimental results that demonstrate the same gene cluster instability in cultured cells that are deficient for the human BLM protein. Bloom’s Syndrome (BS) results from a genetic mutation that results in the abolition of BLM protein, one of human RecQ helicase. Studies of Bloom’s Syndrome have reported a 10-fold increase in sister chromatid exchanges during mitosis which has primarily been attributed to dysregulated homologous recombination. BS also has a strong predisposition to a broad spectrum of malignancies. Biochemical studies have determined that the BLM protein works in conjunction with TOPOIIIα and RMI1/RMI2 to function as a Holliday Junction dissolvase that suppress inadvertent crossover formation in mitotic cells. Because of the similarities in their biochemical activities it was suggested that another DNA helicase found in E. coli, the RecG DNA translocase, is the functional analog of BLM. RecG shares no sequence homology with BLM but it can complement both the sister chromatid exchange elevation and the gene- cluster instability phenotype caused by BLM deficiency. This indicates that the physiological function of BLM that is responsible for these phenotypes rests somewhere in the shared biochemical activities of these two proteins. These data taken together give new insights into the physiological mechanism of BLM protein and the use of Bloom’s Syndrome as a model for carcinogenesis.

La réplication fidèle de l’ADN au cours du cycle cellulaire est essentielle au maintien de l’intégrité du génome à travers les générations. Toutefois, de nombreux éléments peuvent perturber et compromettre la réplication et donc cette intégrité. La mitomycine C (MMC) est une molécule génotoxique utilisée en chimiothérapie. Elle forme des liaisons covalentes entre les deux brins d’ADN, ce qui est un obstacle à la bonne réplication de l’ADN. La rencontre de la fourche de réplication avec une liaison covalente entre les deux brins d’ADN va aboutir à une cassure double brin. Escherichia coli (E.coli) est un modèle d’étude très étendu car facile d’utilisation, permettant d’aborder des notions complexes. E coli possède divers mécanismes pour réparer ces lésions dont le régulon SOS. Le régulon SOS est un ensemble de gènes sous contrôle d’un promoteur réprimé par la protéine LexA. En réponse à des dommages à l’ADN, LexA est dégradé et les gènes du régulon sont activés.En utilisant une technique de biologie moléculaire qui permet de quantifier l’interaction entre deux chromatides sœurs restées cohésives derrière la fourche de réplication (étape appelée cohésion des chromatides sœurs), nous avons montré qu’en réponse à des cassures double brin générées par la MMC, la cohésion entre les chromatides sœurs nouvellement répliquées est maintenue. Ce phénomène est dépendant de RecN, une protéine induite de façon précoce dans le régulon SOS. RecN est une protéine de type SMC (structural maintenance of chromosomes), un groupe de protéines impliqué dans la dynamique et la structure du chromosome. En parallèle, des techniques de microscopie confocale et de marquage du chromosome par des protéines fluorescentes ont permis de montrer que la protéine RecN est impliquée dans une condensation globale du nucléoide suite à un traitement par la MMC. Cette condensation du nucléoide s’accompagne d’un rapprochement des chromatides sœurs ségrégées. Ces deux phénomènes, médiés par RecN pourraient permettre une stabilisation globale des nucléoides et favoriser l’appariement des chromatides sœurs pour permettre la recombinaison homologue.De façon intéressante, l’inhibition de Topoisomérases de type II (Topoisomerase IV et Gyrase) permettent de restaurer le phénotype d’un mutant recN en viabilité et en cohésion des chromatides sœurs. Les Topoisomérases sont des protéines qui prennent en charge les liens topologiques générés par la réplication et la transcription). Les liens topologiques non éliminés par les Topoisomerases permettraient de garder les chromatides sœurs cohésives et favoriser la réparation, même en l’absence de RecN.De plus, une expérience de RNA seq (séquençage de tout le transcriptome de la bactérie) a révélé que dans un mutant recN, le régulon SOS est moins induit que dans les cellules sauvages. Ceci va de pair avec une déstructuration des foci de réparation RecA. Il est possible que le rapprochement des chromatides sœurs médié par RecN permettrait de stabiliser le filament RecA et donc l’induction du SOS.L’ensemble de ces résultats suggère que RecN, une protéine de type SMC, permet de maintenir la cohésion entre les chromatides sœurs nouvellement répliquées, favorisant la réparation de cassures double brins par recombinaison homologue
Maintaining genome integrity through replication is an essential process for the cell cycle. However, many factors can compromise this replication and thus the genome integrity. Mitomycin C is a genotoxic agent that creates a covalent link between the two DNA strands. When the replication fork encounters the DNA crosslink, it breaks and creates a DNA double strand break (DSB). Escherichia coli (E.coli) is a widely used model for studying complex DNA mechanisms. When facing a DNA DSB, E. coli activates the SOS response pathway. The SOS response comprises over 50 genes that are under the control of a LexA-repressed promoter. Upon a DSB induction, RecA, a central protein of the SOS response will trigger the degradation of LexA and all the SOS genes will be expressed.We have developed a novel molecular biology tool that reveals contacts between sister chromatids that are cohesive. It has been shown in the lab (Lesterlin et al. 2012) that during a regular cell cycle, the two newly replicated sister chromatids stay in close contact for 10 to 20 min before segregating to separate cell halves thanks to the action of Topoisomerase IV. This step is called sister chromatid cohesion. We have used this molecular biology tool to study sister chromatid cohesion upon a genotoxic stress induced by mitomycin C (MMC). We have shown that sister chromatid cohesion is maintained and prolonged when the cell is facing a DSB. Moreover, this sister chromatid cohesion is dependent on RecN, an SOS induced structural maintenance of chromosome-like (SMC-like) protein. In the absence of RecN, the proximity between both sister chromatids is lost and this has a deleterious effect on cell viability. By tagging the chromosome with fluorescent proteins, we have revealed that RecN can also mediated a progressive regression of two previously segregated sister chromatids and this is coordinated with a whole nucleoid compaction. Further studies showed that this genome compaction is orderly and is not the result of a random compaction in response to DNA damage.Interestingly, inhibiting TopoIV in a recN mutant fully restores viability and sister chromatid cohesion suggesting that RecN’s action is mainly structural. Preserving cohesion through precatenanes is sufficient to favor repair and cell viability even in the absence of RecN.An RNA-seq experiment in a WT strain and a recN mutant revealed that the whole SOS response is downregulated in a recN mutant. This suggests that RecN may have an effect on the induction of the SOS response and thus RecA filament formation. This is in good agreement with the change in RecA-mcherry foci formation we observed. In the WT strain, the RecA-mcherry foci are defined as described in previous work. However, in the recN, the RecA-mcherry foci seemed to form bundle like structures. These RecA bundles were previsously described by Lesterlin et al. in the particular case of a DSB occurring on a chromatid that has already been segregated from its homolog. This could mean that in the absence of recN, the sister chromatids segregate and RecA forms bundle like structures in order to perform a search for the intact homologous sister chromatid.Altogether, these results reveal that RecN is an essential protein for sister chromatid cohesion upon a genotoxic stress. RecN favors sister chromatid cohesion by preventing their segregation. Through a whole nucleoid rearrangement, RecN mediates sister chromatid regression, favoring DNA repair and cell viability

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.

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

Orientador: Hernandes Faustino de Carvalho
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-05T10:11:12Z (GMT). No. of bitstreams: 1 Peters_Helene_M.pdf: 3147205 bytes, checksum: 29d84ddab25f17a17efc857545126663 (MD5) Previous issue date: 2005
Resumo: A próstata tem merecido crescente atenção devido à maior incidência de câncer prostático e outras afecções do órgão, que resultam do aumento na longevidade dos indivíduos do sexo masculino em todo o mundo. Além disto, o desenvolvimento e crescimento prostático normal apresenta regulação androgênica e está sujeito a uma série de disruptores endócrinos que afetam o seu crescimento e função, assim como predispõem ao desenvolvimento tumoral. Nosso interesse reside principalmente na remodelação prostática seguida à castração e nas interações epitélio estroma que ocorrem neste órgão. Neste trabalho, investigamos a expressão do inibidor de metaloproteinases (MMPs) RECK, em nível de RNAm, procurando correlacioná-Io com o desenvolvimento pós-natal e com a regressão prostática seguida à castração. Para isto, foram utilizadas técnicas de RT-PCR semiquantitativo, Real time RT-PCR e de hibridação in situ,pareados sempre que possível com a expressão do RNAm e com a atividade de algumas MMPs. Os resultados demonstram que o gene RECK é expresso na próstata ventral de ratos, que existe uma significativa redução na sua expressão ao longo do desenvolvimento pós-natal, que há mecanismos diferenciados controlando a expressão dos pares RECKlMMP-2 e MMP-7/MMP-14. Foi observado também um crescente aCÚInulo da forma ativa da MMP-9, conforme o animal se aproxima da idade adulta. Utilizando RT-PCR semiquantitativo, pudemos determinar que o conteúdo relativo do RNAm para o RECK após a castração não muda, embora haja uma inversão no balanço entre a expressão epitelial (células epiteliais) e estromal (células musculares lisas e fibroblastos), nesta situação. No conjunto, os resultados sugerem que o RECK é expresso por diferentes tipos celulares da próstata ventral de ratos, com mecanismos de regulação complexos provavelmente oriundos da existência de diferentes compartimentos no órgão, ao contrário do que se observa para células isoladas
Abstract: The prostate has deserved increasingly attention due to the growing incidence of prostatic cancer and other prostatic diseases, which can be related to the longevity increase of men around the world. Besides, the normal prostatic development is under androgen regulation and as so is subject to a series of endocrine disruptors which affect its growth and function and predisposes to prostate cancer. Our interest resides on the prostatic remodelling following castration and on the epithelial-stromal relationships known to occur in the organ. In this work, we have investigated the expression of the matrix metalloproteinase inhibitor RECK, at the rnRNA leveI, trying to correlate its expression with the post natal prostatic development and regression after castration, using semiquantitative RT-PCR, Real time RT-PCR and in situ hybridization, paralleled with the determination of some MMPs expression and activity. Tbe results demonstrate that RECK is expressed in the rat ventral prostate, that there is a significative reduction in its expression during the post natal development, which is paralleled by the expression of some MMPs and that the mechanisms controling the pairs RECKJMMP-2 and MMP-7/MMP-14 are different. It was also observed an increased proportion of the active form of MMP-9, as the animal approaches adulthood. Using semiquantitative RT-PCR, we could determine that the relative content ofRECK rnRNA remains unchanged by castration, spite detecting an inversion in the balance between the epithelial (epithelial cells) and stromal (smooth muscle cells and fibroblasts) in this situation. Taken together, the results indicate that RECK is expressed by different cell types of the rat ventral prostate, with regulatory mechanisms appearing more complex, likely resulting ftom the existence of different compartments in the organ opposing what was seen for isolated cells
Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural

Gliomas são tumores altamente invasivos, resistentes aos tratamentos disponíveis atualmente e com alta taxa de mortalidade. A superexpressão de RECK na linhagem de glioma humano T98G comprometeu a capacidade das células de migrar e invadir in vitro, com rearranjo do citoesqueleto e alteração na distribuição espacial de FAK fosforilado. Entretanto, o possível mecanismo envolvido na inibição da migração mediada por RECK não foi desvendado. Para estudarmos os mecanismos envolvidos nesta alteração da capacidade migratória, as células T98G foram transfectadas com o vetor plasmidial pCXN2-hRECK (RECK+). A via das integrinas, a atividade de alguns membros da família das RhoGTPases e elementos do citoesqueleto foram avaliados através de imunoblotting, imunomarcação e ensaios de pull-down para as células RECK+ em comparação com células T98G não-transfectadas (WT), células T98G transfectadas com vetor pCXN2 na ausência do gene RECK (vetor) e fibroblastos primários humanos (FF287). Nossos resultados mostram um aumento na expressão de integrina β1 e uma diminuição da fosforilação de FAK no sítio de auto-fosforilação Tyr397 que, juntamente com o aumento das fibras de estresse e a diminuição dos lamelipódios, sugerem um fenótipo menos migratório da célula. Porém, quando avaliada a atividade de Rac1, esta se mostrou aumentada, embora uma das vias de ativação de Rac1 seja através da fosforilação de FAK levando à formação dos lamelipódios. A hipótese é que RECK inibe a quebra das adesões focais que participam do processo de migração, dificultando a mobilidade celular. Como as células continuam recebendo o estímulo para migrar, estas ativam Rac1 através de uma via independente de FAK. Além disso, a imunomarcação de paxilina mostrou um aumento no tamanho das adesões focais nas células RECK+, indicando que RECK pode influenciar nas estruturas responsáveis pelo contato célula-matriz.
Gliomas are highly invasive, treatment-resistant and lethal tumors. Overexpression of RECK in human glioma cell line T98G decreased cell migration and invasion in vitro, lead to cytoskeleton rearrangement and caused changes in phospho-FAK distribution. However, the pathway involved in RECK-mediated inhibition of cell migration has not been elucidated yet. To study the mechanisms by which RECK affects cell motility, T98G cells were transfected with pCXN2-hRECK vector (RECK+). Some proteins involved in the integrin pathway, activity of some proteins of RhoGTPase family and cytoskeleton proteins were analyzed through immunoblotting, immunostaining and pull-down assay in RECK+ cells and compared with non-transfected T98G cells, T98G transfected with pCXN2 without RECK gene and human primary fibroblasts (FF287). Our results showed an increase in integrin β1 expression and a decrease in FAK phosphorylation in the Tyr397 site, which together with the increase of stress fibers and decrease of lamellipodia, suggest a less migratory phenotype. Despite this, Rac1 activity was increased even though one of Rac activation pathways is through phospho-FAK, leading to lamellipodium formation. Our hypotheses is that RECK affects focal adhesion turnover, diminishing cell motility. As cells are still receiving a positive signal to migrate, they activate Rac1 through a FAK-independent pathway. Besides that, paxillin immunostaining showed that focal adhesions are larger in RECK+ cells, indicating that RECK can influence structures related with cell-matrix contact.

Os tumores da cérvice-uterina, que representam uma das principais doenças ginecológicas em mulheres na idade reprodutiva em todo o mundo, estão etiologicamente associados com a infecção pelo papilomavírus humano (HPV). A progressão de uma lesão intraepitelial escamosa de baixo-grau (LSIL) a um carcinoma invasivo de cérvix uterina está acompanhada da degradação da matriz extracelular (MEC) devido à ação progressiva das metaloproteinases de matriz (MMP-2, MMP-9 e MMP-14) no processo de invasão e metástase. Entretanto, o balanço entre as MMPs e seus reguladores como RECK e TIMPs é necessário para controlar esta invasão. O objetivo deste projeto consiste em avaliar a atividade e a expressão das metaloproteinases 2, 9, e 14, e caracterizar a expressão do gene supressor de metástase RECK e do inibidor tecidual de metaloproteinases (TIMP-2), em modelo de queratinócitos humanos infectados com retrovírus recombinantes que expressam os oncogenes E6 e/ou E7 de HPV 16, em culturas cultivadas em monocamada e organotípicas. Para isso, utilizamos ensaios de real-time PCR, zimografia, western blot, imunocitoquímica, ensaio de ELISA e imunohistoquímica. Em culturas em monocamada observamos que as células que expressam as oncoproteínas E6E7 de HPV16 apresentaram menores níveis protéicos de RECK e TIMP-2 em relação ao controle pXLSN. Quando analisamos as culturas organotípicas, também observamos esta diminuição dos níveis de RNAm e protéicos de RECK em rafts que expressam E6E7, acompanhado pelo aumento da atividade de MMP-9, em relação ao controle. Também observamos que o tratamento das culturas com a citocina TNF aumenta a expressão gênica, protéica e atividade de MMP-9 em todas as linhagens analisadas. Além disso, os oncogenes E6 e/ou E7 não afetam a expressão e/ou atividade de MMP-2, MT1-MMP. Nossos dados demonstraram que a expressão das oncoproteínas E6E7 de HPV16 estão relacionadas com o desequilíbrio entre MMPS e seus inibidores, sugerindo que em uma fase pré-invasiva do carcinoma cervical, não somente as MMPs, mas, principalmente seus inibidores são críticos para início da progressão tumoral.
Cervical cancer is etiologically associated with to high-risk human papillomavirus (HPV) infection. It has been observed that matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation during cervical carcinoma progression. Moreover, a counterbalancing among MMPs and their regulators, such as TIMPs and RECK, is necessary to modulate invasion. In order to study the effect of HPV oncogenes on MMPs expression, primary human keratinocytes (PHKs) were infected with recombinant retroviruses expressing wild-type HPV16 E6 and/or E7 oncogenes and were used to seed monolayers and organotypic cultures. Quantitative real-time PCR (Q-PCR), western blot, zimography, immunocitochemistry, ELISA assay and immunohistochemistry were used to determine the expression level and activity of MMP-2, MMP-9, MT1-MMP and their inhibitors RECK and TIMP-2. We observed that cultures expressing E6E7 presented lower RECK and TIMP-2 protein levels than control keratinocytes. In addition, rafts cultures presented the same lower RECK levels additionally presenting higher MMP-9 activity than control. Furthermore, we observed that expression of E6 and/or E7 proteins do not affect MMP-2 and MT1-MMP protein levels and/or activity. We also observed that TNF treatment enhance the MMP-9 gene and protein expression and activity in all studied cell lines. Taken together, our results demonstrate that HPV16E6E7 expression is related with the unbalance between MMPs and their inhibitors, suggesting that in the initial steps of HPV-related cervical disease, not only MMPs but also RECK and TIMP-2 are critical for tumor progression.

La protéine RecA intervient dans la réparation des lésions de l’ADN (i) en clivant le répresseur LexA et en déréprimant les gènes SOS qui sont sous le contrôle de LexA, (ii) en clivant la protéine UmuD dont seule la forme clivée est active dans la réparation fautive, (iii) en participant directement à la recombinaison post-replicative et à la réparation fautive. Nous avons étudié l'importance des différentes activités de la protéine RecA dans la régulation de la réparation SOS grâce à l’emploi de mutants ponctuels. Dans les résultats rapportés dans le chapitre l, l'activité coprotéase de différents mutants recA a été estimée par leur capacité à induire des mutants du phage λ. Ces phages codent pour des répresseurs présentant des sensibilités différentes au clivage protéolytique. Nos résultats montrent que l'activité coprotéase dépend in vivo (i) du nombre de lésions et de l'aptitude de la cellule à éliminer ces lésions (ii) de la quantité de molécules RecA (iii) de la capacité de la protéine RecA à interagir avec les cofacteurs (NTP et ADN simple brin) pour former un complexe actif, (iv) de l'affinité du répresseur pour la protéine RecA. Le chapitre suivant concerne l'interaction de la protéine RecA avec les différentes protéines dont elle stimule le clivage: LexA, UmuD, λcI et Φ80cl. De nouveaux mutants recA ont été isolés. Leur étude montre que le changement d'un acide aminé affecte spécifiquement le clivage de certains répresseurs. En effet, le mutant recA1730 ne clive pas la protéine LexA, le mutant recA1734 ne clive pas UmuD et Φ80cl et le mutant recA430 clive partiellement LexA et pas du tout UmuD et λcI. Bien que les changements dans ces trois protéines ne concernent qu'un acide aminé, ils altèrent également l’activité de recombinaison de la protéine RecA. Deux classes de mutants altérés dans la recombinaison peuvent être distinguées : les mutants dont l'activité de recombinaison est partiellement restaurée par la surproduction de protéine RecA dans des bactéries LexA (Def) et ceux qui sont insensibles à cet effet. Le mutant recA1735 qui est décrit dans le 3ème chapitre présente un phénotype nouveau : la protéine RecA1735 amplifiée dans une bactérie LexA (Def) est létale. La protéine RacA1735 présente une activité coprotéase normale et une activité de recombinaison réduite. Un excès des protéines UmuCD impliquées dans la réparation fautive inhibe l'effet létal de l’amplification de la protéine RecA1735. Nous montrons que l'accumulation de protéine UmuCD réduit l'activité recombinatrice de la protéine RecA+. L'effet létal de la protéine RecA1735 pourrait être dû à des « accidents » au cours de la recombinaison qui causeraient des dommages irréversibles de l'ADN. Le dernier chapitre porte sur la comparaison des gènes PsiB portés par les plasmides F et R6-5. La présence d'un plasmide R6-5 inhibe l'activité coprotéase de la protéine RecA alors qu'aucune inhibition n'est détectée en présence du plasmide F. Bien que les protéines Psi B produites par les deux plasmides soient très semblables (seulement 4 acides diffèrent sur 152) seul le plasmide R6-5 Inhibe l'induction du système SOS. Nous montrons que cette activité anti-SOS est due à la production de protéine PsiB. J'ai résolu le paradoxe des différences d'inhibition par les plasmides F et R6-5 en montrant que les séquences de régulation du gène PsiB diffèrent dans une région de 71 nucléotides comprenant les sites -10 et -35 de reconnaissance de la RNA polymérase. L'activité des deux promoteurs a été mesurée grâce à leur fusion à un fragment du gène lacZ. Le promoteur du gène PsiB du plasmide R6-5 est trois fois plus actif que celui du plasmide F ce qui pourrait expliquer les différences d'inhibition observées.

Tuberculosis, caused by the infection with Mycobacterium tuberculosis remained as a major global health challenge with one third of world population being infected by this pathogen. M. tuberculosis can persist for decades in infected individuals in the latent state as an asymptomatic disease and can emerge to cause active disease at a later stage. Thus, pathways and the mechanisms that are involved in the maintenance of genome integrity appear to be important for M. tuberculosis survival, persistence and pathogenesis. Helicases are ubiquitous enzymes known to play a key role in DNA replication, repair and recombination. However, role of helicases in providing selective advantage for M. tuberculosis survival and genome maintenance is obscure. Therefore, understanding the role of various helicases could provide insights into the M. tuberculosis survival, persistence and pathogenesis in humans. This information could be useful in considering helicases as a novel therapeutic target as well as developing effective vaccines. The research focus of my thesis has been to understand the role of helicases in safeguarding the M. tuberculosis genome from various genotoxic stresses. The major focus of the current study has been addressed towards understanding the role of M. tuberculosis RecG (MtRecG) helicase in recombinational repair and in remodeling stalled replication forks. This study highlights the importance of RecG helicase in the maintenance of genome integrity via DNA repair, recombination and in remodeling the stalled replication forks in M. tuberculosis. The thesis has been divided into following sections as follows: Chapter I: General introduction that describes the causes and consequences of replication stress and DNA repair pathways in M. tuberculosis The genome is susceptible to various types of damage induced by exogenous as well as endogenous DNA damaging agents. Unrepaired or misrepaired DNA lesions can lead to gross chromosomal rearrangements and ultimately cell death. Thus, organisms have evolved with efficient DNA damage response machinery to cope up with deleterious effects of genotoxic agents. Accurate transmission of genetic information requires error-free duplication of chromosomal DNA during every round of cell division. Defects associated with replication are considered as a major source of genome instability in all organisms. Normal DNA replication is hampered when the fork encounters road blocks that have the potential to stall or collapse a replication fork. The types of lesions that potentially block replication fork include lesions on the template DNA, various secondary structures, R-loops, or DNA bound proteins. To understand the DNA damage induced replication stress and the role of fork remodeling enzymes in the repair of stalled replication forks and its restart, chapter I of the thesis has been distributed into multiple sections as follows: Briefly, initial portion of the chapter describes overall replication process in prokaryotes highlighting the importance of coordinated replisome assembly and disassembly during initiation and termination. Later section discusses about various types of exogenous and endogenous DNA damages leading to replication fork stalling. Subsequent section of chapter I provide detailed description and mechanism of various repair pathways cell operates to repair such damages. Chapter I further summarizes causes of stalled replication forks majorly including template lesions, natural impediments like DNA secondary structures and DNA-protein cross links. Subsequent section discusses various pathways of replication restart that include essential role of primosomal proteins in reloading replisome machinery at stalled replication forks. Subsequent section of chapter I provide a comprehensive description of replication fork reversal (RFR) and mechanism of replication restart. RFR involves unwinding of blocked forks via simultaneous unwinding and annealing of parental and daughter strands to generate Holliday junction (HJ) intermediate. Genetic and biochemical studies highlighted the importance of RecG, RuvAB and RecA proteins in driving RFR reaction in E. coli. Hence, in the subsequent chapter, the functional role of RecG, RuvAB and RecA in replication-recombination processes has been discussed. Last section of the chapter devotes completely to M. tuberculosis, its genome dynamics and the various pathways of mycobacterial DNA repair. M. tuberculosis experiences substantial DNA damage inside host macrophages owing to the acidic environment, reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) which are sufficient enough to cause replication stress. To gain insights into the role of M. tuberculosis RecG helicase in DNA repair, recombination and in remodeling the stalled replication forks the following objectives were laid for my PhD thesis: 1 To understand the functional role of M. tuberculosis RecG (MtRecG) in DNA repair and recombination. 2 To investigate the distinct role(s) of MtRecG, MtRuvAB and MtRecA in remodeling the stalled replication forks. Chapter II: Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination In order to survive and replicate in a variety of stressful conditions during its life cycle, M. tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. Helicases are one such ubiquitous enzyme involved in all DNA metabolic transaction pathways for maintenance of genome stability. To understand the role of M. tuberculosis RecG (MtRecG) helicase in recombination and repair, we carried out functional and biochemical studies. In our study, we show that M. tuberculosis RecG expression was induced in response to different genotoxic agents. Strikingly, expression of M. tuberculosis RecG in Escherichia coli ∆recG mutant strain provided protection against MMC, MMS and UV-induced cell death. Purified M. tuberculosis RecG exhibited higher binding affinity for the Holliday junction (HJ) as compared to a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile junction, branch migration and resolution was evident only in the case of the mobile junction. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently as compared to the mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structures, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Altogether, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as in stress conditions. Chapter III: Mycobacterium tuberculosis RecG but not RuvAB or RecA is efficient at remodeling the stalled replication forks: Implications for multiple mechanisms of replication restart in mycobacteria Aberrant DNA replication, defects in the protection and restart of stalled replication forks are a major cause of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen responds to different types of replication stress and DNA damage is unclear. In our study, we show that M. tuberculosis RecG rescues E. coli ∆recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without leading strand ssDNA gap. Interestingly, SSB suppresses the MtRecG and MtRuvAB mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG catalyzed replication fork remodeling and restart pathways in vivo.

Qi Wei.
"November 2003."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2003.
Includes bibliographical references (p. 158-185).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

碩士
國立陽明大學
遺傳學研究所
84
recF，recO和recR基因是原先被發現參與RecF途徑之基因，目前有許多證據顯示此三個基因產物作用於DNA重組的同一步驟。現今有關RecF，RecO和RecR蛋白的在DNA重組途徑中所扮演的角色仍不清楚。 在本論文中，我們針對已純化的RecF，RecO和RecR蛋白的生化功能作進一步之探討，以期瞭解這些蛋白在DNA重組和修復上扮演的角色。主要的工作包括：(1)探討RecF，RecO和RecR與DNA結合之能力；(2)探討RecF，RecO和RecR的helix-unwinding活性。 首先，在RecF，RecO和RecR與DNA結合能力方面，我們證實單獨的RecF或RecO可以與ssDNA結合，而RecR不能。但在有RecO存在下，則RecR可與ssDNA結合。當RecF，RecO和RecR同時與ssDNA作用時，發現這三個蛋白皆可與ssDNA結合。 而在DNA unwinding活性方面，我們發現RecO有明確的活性，RecO的helix-unwinding活性需要有ATP及鎂離子，ATP之需求可以dATP取代，但無法以ATPγS或其它NTP替代，鎂離子之需求可以錳離子取代，但無法以鈣離子或鋅離子取代，RecOunwind helix的能力與RecO蛋白質之濃度成正比，當RecO之量可飽和結合至ssDNA時，其unwilnd DNA之活性最高，最後此活性會受到高濃度之鎂離子或鹽的抑制。根據我們發現RecO具有helix-unwinding活性，認為RecFOR在DNA重組作用中扮演新的角色。在本文中有將有詳細討論。 The recF, recO and recR genes were originally identified as those affecting the RecF pathways of recombination in Escherichia coli. Several lines of genetic evidence suggest that the recF, recO and recR gene products function at the same step of recombination, possibly at an early presynaptic step. The exact role of RecFOR in DNA recombination are not known. In this work, the interactions of RecF, RecO and RecR with ssDNA and the helix-unwinding activities of RecF, RecO and RecR were examined. We observed that single RecF or RecO can bind to ssDNA while single RecR cannot. In the presence of RecO, but not RecF, the RecR was found to associate with ssDNA. When the RecF, RecO and RecR were reacted with ssDNA, all three proteins were found to associate with DNA. With regards to helix-unwinding activity, we observed that RecO possesses such an activity. The helix-unwinding activity of RecO requires the presence of MgCl2 and ATP in the reaction mixture. The requirement of ATP can be substituted by dATP, but cannot be substituted by ATPγS or other NTPs. The requirement of magnesium can be substituted by manganese, but not by calcium or zinc. The unwinding activity is proportional to the concentration of RecO protein and is sensitive to high concentration of MgCl2 or NaCl. Maximal unwinding was observed when the RecO protein to DNA nucleotides was greater than 0.05. Our finding that RecO possesses helix-unwinding activity suggests a new role of ReeFOR in DNA recombination. A model for the possible involvement of RecFOR in DNA recombination is presented.

Homologous recombination is a fundamental cellular process evolved to maintain genomic integrity and to generate genetic diversity. It plays a crucial role in DNA repair, correct segregation of meiotic chromosomes and resumption of the stalled replication forks. In vitro, the homologous recombination pathway is kinetically separable into a four step process involving initiation, homologous pairing, branch migration and junction resolution. The process of pairing and strand exchange between two homologous double-stranded DNA molecules leads to the formation of an intermediate structure called the Holliday junction (HJ). The crucial enzyme involved in this step in bacteria is RecA. In eubacteria, the junction is processed by three proteins, collectively referred to as the RuvABC protein complex. RuvA binds to the HJ, while RuvB, a helicase, binds to the RuvA-HJ complex and pumps the duplex DNA thus facilitating branch migration. The work reported here is concerned with structural studies on mycobacterial RecA and RuvA. X-ray crystallography was used to solve the protein crystal structures. The hanging drop vapour diffusion method was used for crystallization in all cases. X-ray intensity data were collected on a MAR Research imaging plate mounted on a Rigaku RU200 X-ray generator except for two data sets collected using synchrotron radiation. The data were processed mostly using Mosflm and Scala and few data sets were processed using the HKL program suite. The molecular replacement method using programs Phaser and AMoRe was used for structure solution. Structure refinements were carried out using programs CNS and PHENIX. Model building was performed using COOT and O. PROCHECK, MOLPROBITY, ALIGN and NACCESS were used for structure validation and analysis of the refined structures. Mycobacterium smegmatis RecA (MsRecA) and its nucleotide complexes crystallize in three different, but closely related, forms characterized by specific ranges of unit cell dimensions. The six crystals discussed in the earlier part of the thesis and the five reported earlier, all grown under the same or very similar conditions, belong to these three forms, all in space group P61. They include one obtained by reducing the relative humidity around the crystal. In all crystals, RecA monomers form filaments around a 61 screw axis. Thus, the c-dimension of the crystal corresponds to the pitch of the RecA filament. As reported in the case of E.coli RecA, the variation in the pitch among the three forms correlate well with the motion of the C-terminal domain of the RecA monomers with respect to the main domain. The domain motion is compatible with formation of inactive as well as active RecA filaments involving monomers with a fully ordered C-domain. It does not appear to influence the movement upon nucleotide-binding of the switch residue Gln 196, which is believed to provide the trigger for transmitting the effect of nucleotide-binding to the DNA-binding region. Interestingly, partial dehydration of the crystal results in the movement of the residue, in a way similar to that caused by nucleotide-binding. The ordering of the DNA-binding loops L1 and L2, which present an ensemble of conformations, is also unaffected by domain motion. The conformation of loop L2 appears to depend upon nucleotide-binding presumably on account of the movement of the switch residue which forms part of the loop. The conformations of loops L1 and L2 are correlated and have implications to intermolecular communications within the RecA filament. The structures resulting from different orientations of the C-domain and different conformations of the DNA-binding loops appear to represent snapshots of the RecA molecule at different phases of activity and provide insights into the mechanism of action of RecA. Crystal structures of mutants of MsRecA involving changes of Gln 196 from glutamine to alanine, asparagine and glutamic acid, wild type MsRecA and several of their nucleotide complexes were subsequently determined using mostly low temperature and partly room temperature X-ray data. At both the temperatures, nucleotide binding results in a movement of Gln 196 towards the bound nucleotide in the wild type protein. This movement is abolished in the mutants, thus establishing the structural basis for the triggering action of the residue in terms of the size, shape and the chemical nature of the side chain. The 25 crystal structures reported in this thesis, along with the 5 MsRecA structures reported earlier, provide further elaboration of the relation among the pitch of the `inactive´ RecA filament, the orientation of the C-terminal domain with respect to the main domain and the location of the switch residue. The low temperature structures define one extreme of the range of positions the C-domain can occupy. The movement of the C-domain is correlated to those of the LexA binding loop and the loop that connects the main and the N-terminal domains. These elements of molecular plasticity are made use of in the transition to the `active´ filament, as evidenced by the recently reported structures of RecA-DNA complexes. The available structures of RecA resulting from X-ray and electron microscopic studies appear to represent different stages in the trajectory of the allosteric transformations of the RecA filament. This work contributes to the description of the early stages of this trajectory and provides insights into structures relevant to the later stages. The interesting results observed in the case of MsRecA prompted similar studies on the RecA from Mycobacterium tuberculosis (MtRecA). In this study, the crystals were grown at slightly different conditions and examined at different relative humidities and temperatures. Surprisingly, in spite of the 92% sequence identity between the two proteins, the structures indicated MtRecA to be substantially less plastic than MsRecA. The crystal structures do not provide an obvious explanation for this difference. Further studies are warranted to explain the molecular basis of the difference. RuvA, along with RuvB, is involved in branch migration of heteroduplex DNA in homologous recombination. The structures of four crystal forms of RuvA from Mycobacterium tuberculosis (MtRuvA) have been determined. The RuvB-binding domain is cleaved off in one of them. Detailed models of the complexes of octameric RuvA from different species with the Holliday junction have also been constructed. A thorough examination of the structures determined as part of the doctoral programme and those reported earlier bring to light the hitherto unappreciated role of the RuvB-binding domain in determining inter-domain orientation and oligomerization. These structures also permit an exploration of the interspecies variability of structural features such as oligomerization and the conformation of the loop that carries the acidic pin, in terms of amino acid substitutions. These models emphasize the additional role of the RuvB-binding domain in HJ binding. This role along with its role in oligomerization could have important biological implications. In addition to the work on RecA and RuvA, which forms the body of the thesis, the author was also involved in a structural bioinformatics study in which several carbohydrate binding proteins were probed to identify common minimum principles required for binding mannose, glucose and galactose. The study, presented in an Appendix, identified interactions that were specific to particular sugars, leading to individual fingerprints. These fingerprints were then used for exploring lead compounds, using a fragment based approach. This investigation helped the author to familiarize himself with the analysis of protein structures and ligand design based on them.