Tesis sobre el tema "Sister chromatids cohesion"

Afin de préserver leur niche de vie, les bactéries produisent et sécrètent des antibiotiques avec des propriétés génotoxiques. Divers processus moléculaires maintiennent l’intégrité génomique de tous les organismes vivants. Ces évènements sont primordiaux car la structure de l’ADN est endommagée en permanence par le métabolisme cellulaire (tel que le stress oxydatif ou lors de sa réplication), ou d’autres agents de l’environnement. Les antibiotiques sont utilisés pour des applications cliniques afin de traiter les infections ou le cancer. Dans le travail ici présenté, nous analysons la réponse au stress génomique (GSR) induit par deux antibiotiques génotoxiques : la Bléomycine (BLM) et la Mitomycine-C (MMC). Ces deux antibiotiques altèrent l’ADN de différentes manières tout en conduisant à des cassures doubles brins (DSB). Les DSB sont suspectées d’être la cause principale de la mort cellulaire. Les DSB sont réparées par recombinaison homologue (HR). Des études récentes ont révélé que la HR est essentielle pour survivre aux lésions causées par la BLM et la MMC. Les premiers travaux sur le sujet, ainsi que les premiers modèles présentés dans les livres d’éducation, attribuent une voie de réparation particulière, selon le type de lésion à l’ADN. La protéine RecN, induite par le régulons SOS, joue un rôle important dans la réparation de l’ADN et le traitement des lésions générées par ces deux antibiotiques. Cependant, la fonction de RecN dans ces deux processus n’est pas clairement comprise. RecN est une protéine qui joue un rôle dans la maintenance structurelle du chromosome (SMC). Elle se lie sur de l’ADN simple brin (ADNsb) (et qui peut attraper une seconde molécule d’ADN ?). In vitro, RecN stimule la ligation de molécules d’ADN. In vivo, RecN arrête la ségrégation des chromatides soeurs, et induit une compaction extrême du nucléoide. La sur-expression de RecN est toxique pour les cellules et son niveau est régulé par l’enzyme ClpXP, faisant partie du protéasome. RecN interagit avec RecA, toutes deux sont requises pour survivre aux DSB induites par l’endonucléase I-SCE 1. Elles sont généralement associées dans le même groupe épistatique. Des résultats récents suggèrent que RecA et RecN pourraient également avoir des voies d’actions distinctes, toutes deux importantes pour la réparation de l’ADN. Dans l’étude que l’on présente, nous avons utilisé à notre avantage, son implication dans le processus de réparation de deux types de lésions, pour questionner l’implication de RecN dans la réponse génomique au stress (GSR). Nous avons démontré que la dynamique des chromatides soeurs et que le changement de conformation du nucléoïde induit par RecN, est différent selon l’antibiotique utilisé. En présence de lésions induites par la MMC, RecN est requise en pre-traitement des lésions par « réparation par excision de nucléotide » (NER), et son activité sur les chromatides soeurs se manifeste tôt dans le processus de réparation. A l’inverse, en présence de lésions induites par la BLM, l’activité de RecN ne nécessite pas de traitement par le NER, et se manifeste plus tard, durant les phases de récupération. L’analyse par transposition insertion (TIS) a révélé que recN est un des rares gènes du SOS impliqué dans le GSR des deux antibiotiques. L’absence de RecN perturbe grandement le GSR, notamment par l’augmentation de la pression sur le système de réparation par excision de base (BER), tout en réduisant l’importance de la HR. L’analyse du TIS a également mis en évidence, l’implication extrême de multiples voies telles que : les pompes à efflux, la gestion du stress oxydatif, et le contrôle du cycle cellulaire, pour permettre une récupération de l’altération des dommages à l’ADN. L’activité de RecN est un point de bascule entre différentes solutions de réparation. Plus généralement, ce travail illustre que le GSR est un processus intégré que la cellule déploie pour générer les conditions de sa survie
To preserve their niche, bacteria frequently produce and secrete antibiotics with genotoxic properties. Molecular processes that maintain genomic integrity are essential for all organisms. This is necessary because DNA damage can arise during every round of genome duplications. These antibiotics have been used for clinical application to treat infections or cancers. In the present work, we analyzed the Genomic Stress Response (GSR) induced by two genotoxic antibiotics: Bleomycin (BLM) and Mitomycin C (MMC). Although MMC and BLM alter DNA in different ways, they both lead to double strand breaks (DSB). The DSBs are suspected to be the major cause of cell death repaired by homologous recombination (HR). Earlier studies revealed that HR is essential to bacteria to survive BLM and MMC toxicity. Pioneer works and recent textbooks tend to attribute a particular DNA damage response (DDR) to each type of lesions. The RecN protein, induced by the SOS regulon, appeared to play important roles in the processing and repair of DNA lesions generated by MMC and BLM. However, the function of RecN in these two repair processes is not yet understood. RecN is a structural maintenance chromosome (SMC)-like protein that binds on single strand DNA where it can catch a second DNA molecule. In vitro, RecN stimulates the ligation of DNA molecules. In vivo, RecN prevents sister chromatid segregation and promotes an extreme nucleoid compaction. RecN overexpression is toxic for the cell and its level is regulated by ClpXP proteasome. Because RecN interacts with RecA and both are equally required to survive I-SCE 1 mediated DSB, they are generally associated in the same epistatic group. However, recent data suggest that RecA and RecN may also function in genetically distinct pathways, important for the DNA repair. In the present study, we took advantage of RecN involvement in the repair of two different types of DNA lesions to investigate the GSR. We demonstrated that sister chromatid dynamics and nucleoid management by RecN differ according to the drug considered. In presence of MMC-induced lesions, RecN requires a pre-processing of the lesions by the nucleotid excision repair (NER) and its activity on sister chromatids occurs early in the repair process. By contrast, in presence of BLM-induced lesions, RecN activity does not require NER processing and occurs later in the recovery phase. Transposition insertion (TIS) analysis revealed that recN is one of the rare DDR genes involved in the GSR of both drugs. A lack of RecN significantly disturbed the GSR, by increasing notably the pressure on the base excision repair (BER) pathway, while reducing concomitantly the importance of homologous recombination. The TIS analysis also highlighted how important drug tolerance pathways such as: efflux systems, oxidative stress management and cell cycle controllers, are for successful recovery from DNA alterations. Moreover, RecN activity influences the balance between different solutions. More generally, this work illustrates that GSR is an integrated process that cells adopt to create the most appropriate conditions for their survival

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2005.
Title from second page of PDF document. Document formatted into pages; contains [3], vi, 124 p. : ill. Includes bibliographical references.

Le complexe cohésine maintient associées les chromatides sœurs depuis la réplication jusqu’à leur ségrégation en mitose. Une question majeure est de comprendre comment la cohésion est établie lors de la phase S. Chez les mammifères et S. pombe, les cohésines sont associées de manière labile aux chromosomes pré-réplicatifs et l’établissement de la cohésion en phase S s’accompagne de la stabilisation de l’association des cohésines aux chromosomes. L’objectif de ce travail est de comprendre comment la dynamique des cohésines est régulée et comment son inhibition créée la cohésion.En G1 les cohésines associées aux chromosomes s’échangent avec le pool soluble et leur dissociation dépend de Pds5 et Wapl. La première partie de ce travail présente les résultats d’un crible génétique visant à identifier de nouveaux régulateurs de la dynamique des cohésines.L’établissement de la cohésion nécessite l’acétyltransférase Eso1 mais pas en contexte Δwpl1, indiquant que la seule mais essentielle fonction d’Eso1 est de s’opposer à celle de Wapl. L’acétylation de Smc3 par Eso1 contribue mais n’est pas suffisante pour contrecarrer Wapl, suggérant l’existence d’un autre événement dépendant d’Eso1. En G1, Pds5 agit avec Wapl pour dissocier les cohésines des chromosomes mais après la phase S, Pds5 est requise pour leur maintien sur les chromosomes et pour la cohésion à long terme. Pds5 co-localise avec la fraction stable de cohésines mais pas Wapl. Nous suggérons un modèle dans lequel la cohésion est créée par deux événements d’acétylation couplés à la progression de la fourche de réplication conduisant à l’éviction de Wapl des cohésines destinées à produire la cohésion
Following DNA replication, sister chromatids are connected by cohesin to ensure their correct segregation during mitosis. How cohesion is created is still enigmatic. The cohesin subunit Smc3 becomes acetylated by ECO1, a conserved acetyl-transferase, and this change is required for cohesion. As in mammals, fission yeast cohesin is not stably bound to G1 chromosomes but a fraction becomes stable when cohesion is made. The aim of this work was to understand how cohesin dynamics is regulated and how the change in cohesin dynamics creates cohesion.In G1 chromatin bound cohesin exchange with the soluble pool and the unloading reaction relies in part on Wapl. The first part of this study reports on the identification of G1/S factors as new candidate regulators of cohesin dynamics.Following S phase a stable cohesin fraction is made. The acetyl-transferase Eso1 is not required for this reaction when the wpl1 gene is deleted. Yet, it is in wild-type cells, showing that the sole but essential Eso1 function is counteracting Wapl. Eso1 acetylates the cohesin sub-unit Smc3. This renders cohesin less sensitive to Wapl but does not confer the stable binding mode, suggesting the existence of a second Eso1-dependent event. The cohesin sub-unit Pds5 act together with Wapl to promote cohesin removal from G1 chromosomes but after S phase Pds5 is essential for cohesin retention on chromosomes and long term cohesion. Pds5 co-localizes with the stable cohesin fraction whereas Wapl does not. We suggest a model in which cohesion establishment is made by two acetylation events coupled to fork progression leading to Wapl eviction while keeping Pds5 on cohesin complexes intended to make cohesion

Els anells de cohesina, formats per les proteïnes Smc1, Smc3, Scc1 i Scc3, s’uneixen topològicament al DNA, mantenint les parelles de cromàtides germanes unides des de la duplicació del DNA fins al començament de l’anafase. Aquesta funció, coneguda com a Cohesió entre Cromàtides Germanes, permet la biorientació dels cromosomes en el fus mitòtic i, posteriorment, la seva correcta segregació. Es tracta per tant d’una funció fonamental per a la vida. La cohesió entre cromàtides germanes també té altres funcions, com ara afavorir la reparació del dany en el DNA a través de recombinació homòloga. És per aquests motius que la cohesina està sotmesa a diferents nivells de regulació al llarg del cicle cel·lular, a través de diversos factors reguladors i modificacions post-traduccionals. Per exemple, l’acetilació de la subunitat Smc3 és necessària per a que els anells es mantinguin establement units a cromatina. Alteracions en la molècula de cohesina o en la seva regulació poden provocar el desenvolupament de patologies i contribuir en la progressió tumoral. En aquest estudi, descrivim la sumoilació de la cohesina com una nova modificació post-traduccional necessària per la cohesió en Saccharomyces cerevisiae. La sumoilació de la cohesina depèn, en part, de la SUMO lligasa Nse2 i d’un complex Smc5/6 plenament funcional. Totes les subunitats del complex cohesina es sumoilen in vivo durant la replicació del DNA, després de la formació dels anells de cohesina i del seu reclutament a cromatina, en un procés depenent de la unió d’ATP a les subunitats SMC, i independent de l’acetilació de Smc3. Per tal d’alterar l’estat de sumoilació dels anells de cohesina i identificar la rellevància funcional d’aquesta modificació, hem dissenyat un nou sistema experimental que permet eliminar SUMO de totes les proteïnes del complex, basat en la fusió del domini SUMO peptidasa de Ulp1 (UD) a la proteïna Scc1. Les fusions Scc1-UD s’incorporen als anells de cohesina, es carreguen en la cromatina i es localitzen adequadament sobre els cromosomes de llevat. Tanmateix, la desumoilació dels anells de cohesina bloqueja la cohesió entre les cromàtides germanes, aturant el cicle cel·lular en G2/M i provocant la pèrdua de viabilitat de les cèl·lules. Aquests efectes són deguts a l’activitat del domini SUMO peptidasa, i no a problemes estructurals en la proteïna de fusió Scc1-UD, ja que la mutació puntual del centre catalític de UD restaura la cohesió i la viabilitat cel·lular. Experiments en paral·lel suggereixen que la sumoilació de la cohesina podria tenir funcions similars en cèl·lules humanes. Sorprenentment, els anells de cohesina continuen acetilats en absència de sumoilació. Donat que els models actuals proposen que els anells es tanquen de forma estable en ser acetilats, és probable que en absència de sumoilació la cohesina encercli una sola cromàtida. Per tant, proposem que la sumoilació de la cohesina seria necessària durant la replicació del DNA per atrapar les dues cromàtides germanes de forma estable en l’interior de l’anell.
Los anillos de cohesina, formados por las proteínas Smc1, SMC3, Scc1 y Scc3, se unen topológicamente al DNA, manteniendo las parejas de cromátidas hermanas unidas desde la duplicación del DNA hasta el comienzo de la anafase. Esta función, conocida como Cohesión entre Cromátidas Hermanas, permite la biorientación de los cromosomas en el huso mitótico y, posteriormente, su correcta segregación. Se trata por lo tanto de una función fundamental para la vida. La cohesión entre cromátidas hermanas también tiene otras funciones, como favorecer la reparación del daño en el DNA a través de recombinación homóloga. Es por estos motivos que la cohesina está sometida a varios niveles de regulación a lo largo del ciclo celular, a través de diferentes factores reguladores y modificaciones post-traduccionales. Por ejemplo, la acetilación de la subunidad Smc3 es necesaria para que los anillos se mantengan establemente unidos a cromatina. Alteraciones en la molécula de cohesina y/o en su regulación pueden provocar el desarrollo de patologías y contribuir a la progresión tumoral. En este estudio, describimos la sumoilación de la cohesina como una nueva modificación post-traduccional necesaria para la cohesión en Saccharomyces cerevisiae. La sumoilación de la cohesina depende, en parte, de la SUMO ligasa Nse2 y de un complejo Smc5/6 plenamente funcional. Todas las subunidades del complejo cohesina se sumoilan in vivo durante la replicación del ADN, después de la formación de los anillos de cohesina y de su reclutamiento en cromatina, en un proceso dependiente de la unión de ATP a las subunidades SMC, e independiente de la acetilación de Smc3. Con el fin de alterar el estado de sumoilación de los anillos de cohesina e identificar la relevancia funcional de esta modificación, hemos diseñado una nueva aproximación experimental que permite eliminar SUMO de todas las proteínas del complejo, basado en la fusión del dominio SUMO peptidasa de Ulp1 (UD) a la proteína Scc1. Las fusiones Scc1-UD se incorporan a los anillos de cohesina, se cargan en la cromatina y se localizan adecuadamente sobre los cromosomas de levadura. Sin embargo, la desumoilación de los anillos de cohesina impide la cohesión entre las cromátidas hermanas, deteniendo el ciclo celular en G2/M y provocando la pérdida de viabilidad de las células. Estos efectos son debidos a la actividad del dominio SUMO peptidasa, y no a problemas estructurales en la proteína de fusión Scc1-UD, ya que la mutación puntual del centro catalítico de UD restaura la cohesión y la viabilidad celular. Experimentos en paralelo sugieren que la sumoilació de la cohesina podría tener funciones similares en células humanas. Sorprendentemente, los anillos de cohesina continúan acetilados en ausencia de sumoilación. Dado que los modelos actuales proponen que los anillos se cierran establemente al ser acetilados, es probable que en ausencia de sumoilación la cohesina se cierre en torno a una sola cromátida. En consecuencia, proponemos que la sumoilación de la cohesina sería necesaria durante la replicación del ADN para atrapar las dos cromátidas hermanas de forma estable en el interior del anillo.
Cohesin rings composed of the Smc1, Smc3, Scc1 and Scc3 proteins topologically bind to DNA, keeping pairs of sister chromatids together from the time of DNA replication until the onset of anaphase. This feature, known as Sister Chromatid Cohesion (SCC), allows the biorientation of chromosomes on the mitotic spindle, and their subsequent segregation. Sister Chromatid Cohesion also has other roles, such as enabling repair of DNA damage through homologous recombination. Thus, it is not surprising that cohesin is subjected to multiple levels of control during the cell cycle by different regulatory factors and post-translational modifications. For example, acetylation of the Smc3 subunit is required to prevent the opening of cohesin rings, keeping them stably bound to chromatin. Alterations in the cohesin molecule itself and/or its regulation may lead to the development of serious pathologies and can contribute to tumor progression. In this study, we describe the sumoylation of cohesin as a new post-translational modification required for Sister Chromatid Cohesion in Saccharomyces cerevisiae. Sumoylation of cohesin is partially dependent on the Nse2 SUMO ligase and the Smc5/6 complex. All subunits of the cohesin complex are sumoylated in vivo during DNA replication, after the formation of cohesin rings and their recruitment onto chromatin, in a process dependent on the binding of ATP to the SMC subunits, and independent of Smc3 acetylation. In order to alter the sumoylation status of cohesin rings and to identify its functional relevance, we designed a new approach to remove SUMO from all cohesin subunits, based on the fusion of the SUMO peptidase domain of Ulp1 (UD) to the Scc1 protein. Scc1-UD fusions are properly incorporated into cohesin rings, loaded onto chromatin and located along yeast chromosomes. However, desumoylation of cohesin rings prevents Sister Chromatid Cohesion, arresting cells in G2/M and causing the loss of cell viability. These effects are due to the activity of the SUMO peptidase domain rather than structural problems in the Scc1-UD fusion, since mutation of the catalytic site in the UD restores cohesion and cell viability. Parallel experiments suggest that sumoylation of cohesin might have similar functions in human cells. Surprisingly, cohesin rings remain acetylated in the absence of sumoylation. Current models propose that cohesin rings are stably locked once they are acetylated. Therefore, it is likely that in the absence of sumoylation cohesin encircles a single chromatid. Consequently, we propose that sumoylation of cohesin is required during DNA replication to entrap the two sister chromatids inside its ring structure.

Establishment of sister chromatid cohesion is a process thought to occur as the replication fork passes chromosomal loci bound by the cohesin complex. After fork passage, cohesin holds together pairs of replication products to allow their recognition by the mitotic machinery for segregation into daughter cells. In budding yeast, cohesin is loaded onto chromosomes during the G1 phase of the cell cycle. During S phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to promote the establishment of sister chromatid cohesion. At the time of anaphase, Smc3 loses its acetylation again, but the Smc3 deacetylase and the possible importance of Smc3 deacetylation were unknown. I show that the class I histone deacetylase family member Hos1 is responsible for Smc3 deacetylation. Cohesin is protected from deacetylation while bound to chromosomes but is deacetylated as soon as it dissociates from chromosomes at anaphase onset. Nonacetylated Smc3 is required as a substrate for cohesion establishment in the following cell cycle. Smc3 acetylation during DNA replication renders cohesin resistant against the cohesin destabiliser Wapl. However, because in the absence of Wapl cohesin acetylation is dispensable for cohesion establishment, I have turned my attention to additional ‘cohesion establishment factors’ replication fork-associated proteins required for efficient cohesion establishment. These include Chl1, Ctf4, Ctf18, Mrc1, Tof1 and Csm3. I have used genetic and molecular assays to investigate the relationship of these cohesion establishment factors with the cohesin acetylation pathway. This revealed a contribution of all of these factors to efficient cohesin acetylation. However, removal of the cohesin destabiliser Wapl corrected the cohesion defect in all of the cohesion establishment mutants, except Ctf4 and Chl1. Furthermore, my genetic analysis revealed pronounced synthetic interactions of these two factors with Eco1. Ctf4 and Chl1 therefore define a subset of Eco1-independent cohesion establishment factors, whose possible mechanism of action I have started to investigate.

The ‘endless forms most beautiful’ that populate our planet rely on the process of cell division to ensure equal segregation of the cellular content including the DNA to the two daughter cells. The accurate segregation of chromosomes in eukaryotes relies on connection between replicated sister chromatids, a phenomenon known as sister chromatid cohesion. Sister chromatid cohesion is mediated by a conserved ring-like protein complex known as cohesin. Defects in this process can promote aneuploidy and contribute to meiotic segregation errors with adverse consequences for developing embryos. Despite numerous advances into understanding cell division at the molecular level, we still lack a comprehensive list of the participating proteins and complexes. The aim of this thesis was to use available functional genomic and proteomic data to identify novel regulators of mitosis in human cells. Using an RNAi approach, we identified a set of factors involved in pre-mRNA splicing whose depletion prevents successful cell division. Loss of these splicing factors leads to a failure in chromosome alignment and to a protracted mitotic arrest that is dependent on the spindle assembly checkpoint. This mitotic phenotype was accompanied by a dramatic loss of sister chromatid cohesion that we could show happens as soon as DNA replication. While depletion of pre-mRNA splicing mediators had no effect on cohesin loading onto chromatin, it prevented the stable association of cohesin with chromatin. Immunoblotting revealed that the depletion of splicing factors caused a 5-fold reduction in the protein levels of Sororin, a protein required for stable association of cohesin with chromatin in post-replicative cells. Further analysis suggests erroneous splicing of Sororin pre-mRNA upon depletion of splicing factors. Importantly, the sister chromatid cohesion loss caused by depletion of splicing factors could be suppressed by a Sororin transgene that lacks introns. Our results suggest that that pre-mRNA splicing of Sororin is a rate-limiting step in the maintenance of sister chromatid cohesion in human cells. Our work reveals that a primary cellular pathology of compromised pre-mRNA splicing is a mitotic arrest accompanied by split sister chromatids. Our work linking splicing and sister chromatid cohesion has implications for the pathology of Chronic Lymphocytic Leukemia (CLL). One of the splicing factors that we implicate in sister chromatid cohesion is SF3B1, whose gene is one of the most frequently mutated genetic drivers found in CLL patients.

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

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

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.
Includes bibliographical references.
In cell division, the proper segregation of chromosomes requires sister-chromatid cohesion. This physical attachment between sister chromatids is established during DNA replication, maintained throughout mitosis and released at the metaphase-anaphase transition. In meiosis, sister-chromatid cohesion is released along the chromosome arms in the first meiotic division, but retained at the centromere until the second meiotic division. In this thesis, we have analyzed the localization of two cohesion proteins in Drosophila, MEI-S332 and RAD21. MEI-S332 localizes specifically to the centromere from prometaphase to the metaphase-anaphase transition in mitosis, and from prometaphase I to the metaphase II-anaphase II transition in meiosis. We find that the termini of MEI-S332 are required for its localization to chromosomes; these are also the regions that have homology to MEI-S332-like proteins in other organisms. The localization of MEI-S332 does not require the presence of cohesin, an evolutionarily conserved protein complex that is essential for the establishment and maintenance of cohesion, nor a replicated sister chromatid. However, MEI-S332 delocalization is dependent upon the activity of the separase pathway that regulates cohesin release. We have identified and characterized a key subunit of cohesin in Drosophila, DRAD21, and studied its localization in early stages of meiosis in spermatocytes.
(cont.) DRAD21 is nuclear in prophase I, but is not visibly localized on chromosomes in later stages. Although DRAD21 is concentrated in centromeric regions after prometaphase in mitosis, MEI- S332 and DRAD21 do not physically interact in a complex in whole embryo extracts. Immunostaining of spread metaphase chromosomes for MEI-S332 and DRAD21 reveals that the two proteins are not localized to the same domains.
by Janice Ying Lee.
Ph.D.

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Biomedical and Life Sciences (IBLS), University of Glasgow, 2008. Includes bibliographical references. Print version also available.

DNA damage tolerance (DDT) mechanisms are crucial for genome integrity as they allow efficient bypass of endogenously or exogenously generated lesions. Error-free bypass of lesions is accomplished by a recombination-related mechanism, generally referred to as template switching (TS), that allows the recovery of the damaged information from the sister chromatid. Pioneer studies revealed key enzymatic functions required for error-free DDT and identified sister chromatid junctions (SCJs) as crucial DNA intermediates mediating this process. However, little is known on the temporal window and the chromatin/topological context in which TS takes place. Using S. cerevisiae as a model system, here I investigated the contribution of different replication-related processes to recombination-mediated DDT. We identified sister chromatid cohesion and replication-related repriming/primer processing as novel pathways implicated in TS. Unexpectedly, repriming during replication impinges on sister chromatid cohesion, but these two processes differentially contribute to error-free DDT. Repriming activities and other processes influencing the size of the ssDNA gap and the availability of the 5’ end positively influence error-free DDT both in spontaneous and genotoxic stress conditions. Our results provide evidence that error-free DDT is largely a recombination-mediated gap-filling process with different requirements from the recombination mechanism involved in double strand break repair, both in what regards the influence of DNA end processing on the subsequent homology search and strand invasion, and the contribution of the chromatin structural context mediated by sister chromatid cohesion. Our findings suggest that mechanisms promoting damage-bypass via replication-associated SCJ formation affect DDT pathway choice and the establishment of postreplicative sister chromatid synapses.

Chez les bactéries, la ségrégation du chromosome est initiée durant la phase de réplication. Des expériences de time lapse, utilisées pour observer que la dynamique des loci frères durant le cycle cellulaire, montrent que, chez Escherichia coli, les régions sœurs restent colocalisées pour une période significative dans les régions des macrodomaines du chromosome et pour une courte période dans les régions non-structurées. Nous nous sommes posés la question suivante: est ce que l'étape de colocalisation révèle une réelle cohésion entre les chromatides sœurs ? Pour y répondre, nous avons développé un outil génétique, alternatif aux outils de biologie cellulaire, permettant de mesurer la distance entre les chromatides sœurs de manière directe. La fréquence de recombinaison intermoléculaire médiée par la recombinase Cre entre les sites loxP positionnés sur les chromatides sœurs est mesurée pour différentes positions. De cette fréquence, nous avons pu déduire la proximité entre les chromatides sœurs. Nous révélons que les loci frères restent proche l'un de l'autre pour une courte période après la réplication. Nous appelons cette étape la cohésion moléculaire, celle-ci est dépendante du locus considéré. Nous montrons que les facteurs qui favorisent la colocalisation des foci frères n'augmentent pas nécessairement l'habilité des loci frères à recombiner. En effet, la protéine MatP, un acteur de la colocalisation des macrodomaines Ter, n'affecte pas la cohésion entre les deux copies de cette région. La Topoisomérase IV est un facteur essentiel à la ségrégation des chromosomes. En son absence, les chromosomes ne peuvent se ségréger et restent colocalisés dans la cellule. Nous révélons par le test de recombinaison que l'absence de Topoisométase IV dans les cellules provoque une augmentation des interactions entre chromatides sœurs. Au final, nous avons montré que l'étape de cohésion est différente de la colocalisation, que les mécanismes moléculaires diffèrent d'une étape à l'autre et que les liens de précaténation moduleraient la cohésion post-réplicative entre chromatides sœurs.

Cell division is a highly dynamic process where sister chromatids remain associated with each other from the moment of DNA replication until the later stages of mitosis, giving rise to two daughter cells with equal genomes. The “molecular glue” that links sister DNA molecules is called cohesin, a tripartite ring-like protein complex composed of two Structural Maintenance of Chromosome proteins (Smc1 and Smc3) bridged by a kleisin subunit Rad21/Scc1, that together prevent precocious sister chromatid separation. Accumulating evidence has suggested that cohesion decay may be the cause of segregation errors that underlie certain human pathologies. However it remains to be determined how much cohesin loss abolishes functional sister chromatid cohesion. To answer these questions, we have developed different experimental conditions aiming to titrate the levels of cohesin on mitotic chromosomes in a precise manner. Using these tools, we will determine the minimal amount of cohesin needed to confer functional cohesion. The approaches described here take advantage of a system in Drosophila melanogaster where the Tobacco Etch Virus (TEV) protease can cleave the Rad21 subunit of cohesin leading to precocious sister chromatid separation. Firstly, we tried to express different levels of TEV protease to obtain partial loss of cohesion. However, this approach has failed to produce systematic different levels of sister chromatid separation. Most of the work was therefore focused on a second strategy, for which we established strains with different levels of cohesin sensitive/cohesin resistant to TEV protease. Strains containing different amounts of functional cohesin (TEV resistant) were tested by in vitro cleavage and by in vivo injections in embryos for their ability to promote sister chromatid cohesion. Our results reveal that removal of half of the cohesin complexes does not impair chromosome segregation, implying that chromosome cohesion is less sensitive to cohesin amounts than previously anticipated.

The accurate and efficient dissemination of replicated chromosomes into daughter cells is fundamental to all aspects of biology. Chromosomal missegregation can lead to aneuploid chromosome configurations which are a hallmark of cancer cells and also a leading cause of birth defects and infertility in humans. Given that chromosome missegregation can result in such disastrous consequences, cells have evolved mechanisms to ensure the faithful segregation of chromosomes, one of which is sister-chromatid cohesion which is mediated by the cohesin complex. Cohesin is a multi-protein complex thought to be the primary effector of sister chromatid cohesion in all eukaryotes. In yeast, cohesin is loaded onto chromosome arms in S-phase where it maintains sister chromatid cohesion until the metaphaseanaphase transition. Sister chromatid separation is then triggered by the site-specific cleavage of the RAD21 cohesin subunit. In metazoan species, including Drosophila, the bulk of cohesin dissociates form chromosomes in prophase, leaving a minor pool of centromere-associated cohesin to maintain sister-chromatid cohesion until anaphase. Exactly how the various cohesin subunits and their regulators orchestrate these events has yet to be fully elucidated. Meiotic cohesin complexes are subjected to additional levels of regulation to accommodate the different types of cell division that occur to produce haploid gametes. In humans, premature loss of meiotic sister chromatid cohesion has been proposed as the most likely molecular cause for sporadic aneuploidy linked to advanced maternal age. The results presented in this thesis begin with a description of the rationale and approach used to identify the DRAD21 separase cleavage sites, and subsequently mutate them using site-directed mutagenesis. Characterisation of the dominant alleles generated is described, as is the first evidence of DRAD21 proteolysis. The over expression of non-cleavable DRAD21 isoforms was investigated in a range of different tissues and developmental stages, and was shown to dominantly reduce the size of adult tissue. These data suggested that overexpression of non-cleavable isoforms of DRAD21 in dividing cells increased levels of cell death. Analysis of the cellular effects of non-cleavable DRAD21 overexpression in the developing eye imaginal disc confirmed that the level of apoptosis was increased in cells expressing non-cleavable DRAD21, and that this DRAD21 isoform induced mitotic delay or arrest, consistent with a defect in mitotic progression. This is the first description of a Drad21 mutant phenotype. The reduced and roughened eye phenotype generated as a result of non-cleavable DRAD21 expression in the eye imaginal discs provided a tool to use in genetic studies of DRAD21 function. Genetic analysis showed that known and predicted cohesin regulators are capable of modulating the DRAD21 eye phenotype, therefore establishing the suitability of this phenotype for use in a genetic screen. The entire Drosophila genome was screened for genetic modifiers of the DRAD21 eye phenotype. In total 62 interacting genomic regions were identified, spanning chromosomes two, three and four. Analysis of these interactions revealed both enhancers and suppressors of the DRAD21 eye phenotype, and genetic dissection of some of the interacting regions allowed 13 modifier loci to be unequivocally identified at the molecular level. Specifically, ten distinct interacting regions were examined at the molecular level and ten suppressor loci and three enhancer loci were identified and a mechanism by which these interactions may be occurring was proposed for each. These studies are likely to significantly influence our current understanding of metazoan chromosome dynamics and identify novel regulators of chromosome segregation. To date, some interacting loci identified at the molecular level have established roles in chromosome cohesion, while for others this study provides the first evidence for their role in this process. These studies will identify both novel regulators of chromosome segregation and hopefully provide a shortlist of genes that when functionally impaired may incrementally increase the risk of chromosome missegregation and aneuploidy in humans.