Literatura académica sobre el tema "Sister chromatids cohesion"

Cells delayed in metaphase with intact mitotic spindles undergo cohesion fatigue, where sister chromatids separate asynchronously, while cells remain in mitosis. Cohesion fatigue requires release of sister chromatid cohesion. However, the pathways that breach sister chromatid cohesion during cohesion fatigue remain unknown. Using moderate-salt buffers to remove loosely bound chromatin cohesin, we show that “cohesive” cohesin is not released during chromatid separation during cohesion fatigue. Using a regulated protein heterodimerization system to lock different cohesin ring interfaces at specific times in mitosis, we show that the Wapl-mediated pathway of cohesin release is not required for cohesion fatigue. By manipulating microtubule stability and cohesin complex integrity in cell lines with varying sensitivity to cohesion fatigue, we show that rates of cohesion fatigue reflect a dynamic balance between spindle pulling forces and resistance to separation by interchromatid cohesion. Finally, while massive separation of chromatids in cohesion fatigue likely produces inviable cell progeny, we find that short metaphase delays, leading to partial chromatid separation, predispose cells to chromosome missegregation. Thus, complete separation of one or a few chromosomes and/or partial separation of sister chromatids may be an unrecognized but common source of chromosome instability that perpetuates the evolution of malignant cells in cancer.

Sister chromatid cohesion is essential for tension-sensing mechanisms that monitor bipolar attachment of replicated chromatids in metaphase. Cohesion is mediated by the association of cohesins along the length of sister chromatid arms. In contrast, centromeric cohesin generates intrastrand cohesion and sister centromeres, while highly cohesin enriched, are separated by >800 nm at metaphase in yeast. Removal of cohesin is necessary for sister chromatid separation during anaphase, and this is regulated by evolutionarily conserved polo-like kinase (Cdc5 in yeast, Plk1 in humans). Here we address how high levels of cohesins at centromeric chromatin are removed. Cdc5 associates with centromeric chromatin and cohesin-associated regions. Maximum enrichment of Cdc5 in centromeric chromatin occurs during the metaphase-to-anaphase transition and coincides with the removal of chromosome-associated cohesin. Cdc5 interacts with cohesin in vivo, and cohesin is required for association of Cdc5 at centromeric chromatin. Cohesin removal from centromeric chromatin requires Cdc5 but removal at distal chromosomal arm sites does not. Our results define a novel role for Cdc5 in regulating removal of centromeric cohesins and faithful chromosome segregation.

Sister-chromatid cohesion, thought to be primarily mediated by the cohesin complex, is essential for chromosome segregation. The forces holding the two sisters resist the tendency of microtubules to prematurely pull sister DNAs apart and thereby prevent random segregation of the genome during mitosis, and consequent aneuploidy. By counteracting the spindle pulling forces, cohesion between the two sisters generates the tension necessary to stabilize microtubule–kinetochore attachments. Upon entry into anaphase, however, the linkages that hold the two sister DNAs must be rapidly destroyed to allow physical separation of chromatids. Anaphase cells must therefore possess mechanisms that ensure faithful segregation of single chromatids that are now attached stably to the spindle in a manner no longer dependent on tension. In the present review, we discuss the nature of the cohesive forces that hold sister chromatids together, the mechanisms that trigger their physical separation, and the anaphase-specific changes that ensure proper segregation of single chromatids during the later stages of mitosis.

Faithful genome transmission in dividing cells requires that the two copies of each chromosome’s DNA package into separate but physically linked sister chromatids. The linkage between sister chromatids is mediated by cohesin, yet where sister chromatids are linked and how they resolve during cell cycle progression has remained unclear. In this study, we investigated sister chromatid organization in live human cells using dCas9-mEGFP labeling of endogenous genomic loci. We detected substantial sister locus separation during G2 phase irrespective of the proximity to cohesin enrichment sites. Almost all sister loci separated within a few hours after their respective replication and then rapidly equilibrated their average distances within dynamic chromatin polymers. Our findings explain why the topology of sister chromatid resolution in G2 largely reflects the DNA replication program. Furthermore, these data suggest that cohesin enrichment sites are not persistent cohesive sites in human cells. Rather, cohesion might occur at variable genomic positions within the cell population.

Cohesin associates with distinct sites on chromosomes to mediate sister chromatid cohesion. Single cohesin complexes are thought to bind by encircling both sister chromatids in a topological embrace. Transcriptionally repressed chromosomal domains in the yeastSaccharomyces cerevisiaerepresent specialized sites of cohesion where cohesin binds silent chromatin in a Sir2-dependent fashion. In this study, we investigated the molecular basis for Sir2-mediated cohesion. We identified a cluster of charged surface residues of Sir2, collectively termed the EKDK motif, that are required for cohesin function. In addition, we demonstrated that Esc8, a Sir2-interacting factor, is also required for silent chromatin cohesion. Esc8 was previously shown to associate with Isw1, the enzymatic core of ISW1 chromatin remodelers, to form a variant of the ISW1a chromatin remodeling complex. WhenESC8was deleted or the EKDK motif was mutated, cohesin binding at silenced chromatin domains persisted but cohesion of the domains was abolished. The data are not consistent with cohesin embracing both sister chromatids within silent chromatin domains. Transcriptional silencing remains largely intact in strains lackingESC8or bearing EKDK mutations, indicating that silencing and cohesion are separable functions of Sir2 and silent chromatin.

The cohesin complex facilitates faithful chromosome segregation by pairing the sister chromatids after DNA replication until mitosis. In addition, cohesin contributes to proficient and error-free DNA replication. Replisome progression and establishment of sister chromatid cohesion are intimately intertwined processes. Here, we review how the key factors in DNA replication and cohesion establishment cooperate in unperturbed conditions and during DNA replication stress. We discuss the detailed molecular mechanisms of cohesin recruitment and the entrapment of replicated sister chromatids at the replisome, the subsequent stabilization of sister chromatid cohesion via SMC3 acetylation, as well as the role and regulation of cohesin in the response to DNA replication stress.

Sister chromatid cohesion is essential to maintain stable connections between homologues and sister chromatids during meiosis and to establish correct centromere orientation patterns on the meiosis I and II spindles. However, the meiotic cohesion apparatus in Drosophila melanogaster remains largely uncharacterized. We describe a novel protein, sisters on the loose (SOLO), which is essential for meiotic cohesion in Drosophila. In solo mutants, sister centromeres separate before prometaphase I, disrupting meiosis I centromere orientation and causing nondisjunction of both homologous and sister chromatids. Centromeric foci of the cohesin protein SMC1 are absent in solo mutants at all meiotic stages. SOLO and SMC1 colocalize to meiotic centromeres from early prophase I until anaphase II in wild-type males, but both proteins disappear prematurely at anaphase I in mutants for mei-S332, which encodes the Drosophila homologue of the cohesin protector protein shugoshin. The solo mutant phenotypes and the localization patterns of SOLO and SMC1 indicate that they function together to maintain sister chromatid cohesion in Drosophila meiosis.

In meiosis, a physical attachment, or cohesion, between the centromeres of the sister chromatids is retained until their separation at anaphase II. This cohesion is essential for ensuring accurate segregation of the sister chromatids in meiosis II and avoiding aneuploidy, a condition that can lead to prenatal lethality or birth defects. The Drosophila MEI-S332 protein localizes to centromeres when sister chromatids are attached in mitosis and meiosis, and it is required to maintain cohesion at the centromeres after cohesion along the sister chromatid arms is lost at the metaphase I/anaphase I transition. MEI-S332 is the founding member of a family of proteins that protect centromeric cohesion but whose members also affect kinetochore behaviour and spindle microtubule dynamics. We compare the Drosophila MEI-S332 family members, evaluate the role of MEI-S332 in mitosis and meiosis I, and discuss the regulation of localization of MEI-S332 to the centromere and its dissociation at anaphase. We analyse the relationship between MEI-S332 and cohesin, a protein complex that is also necessary for sister-chromatid cohesion in mitosis and meiosis. In mitosis, centromere localization of MEI-S332 is not dependent upon the cohesin complex, and cohesin retains its association with mitotic chromosomes even in the absence of MEI-S332.

During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.

Several lines of evidence suggest the existence in the eukaryotic cells of a tight, yet largely unexplored, connection between DNA replication and sister chromatid cohesion. Tethering of newly duplicated chromatids is mediated by cohesin, an evolutionarily conserved hetero-tetrameric protein complex that has a ring-like structure and is believed to encircle DNA. Cohesin is loaded onto chromatin in telophase/G1 and converted into a cohesive state during the subsequent S phase, a process known as cohesion establishment. Many studies have revealed that down-regulation of a number of DNA replication factors gives rise to chromosomal cohesion defects, suggesting that they play critical roles in cohesion establishment. Conversely, loss of cohesin subunits (and/or regulators) has been found to alter DNA replication fork dynamics. A critical step of the cohesion establishment process consists in cohesin acetylation, a modification accomplished by dedicated acetyltransferases that operate at the replication forks. Defects in cohesion establishment give rise to chromosome mis-segregation and aneuploidy, phenotypes frequently observed in pre-cancerous and cancerous cells. Herein, we will review our present knowledge of the molecular mechanisms underlying the functional link between DNA replication and cohesion establishment, a phenomenon that is unique to the eukaryotic organisms.

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.

Abstract The goal of cell division is to transmit the genome from one generation to the next. This occurs in two essential steps: the genome is first replicated in S phase and then during mitosis the two copies are separated and transported into separate cytoplasmic domains destined to become new cells. This transport function is specified primarily by the centromeres, specialized nucleoprotein domains present in a single copy on each chromosome (1). Centromeres direct assembly of the machinery for microtubule binding and motor activities, known as the kinetochore, at the surface of each sister chromatid, maintain cohesion between sister chromatids, and possess regulatory elements that integrate chromosome motility and spindle function with cell cycle control pathways. During mitosis the kinetochores interact with spindle microtubules first to bind the spindle in prometaphase, then to achieve the crucial bipolar orientation in metaphase and finally to drive poleward movement in anaphase. A parallel system of regulatory elements located within the centromere monitors kinetochore attachment and communicates with the spindle to control anaphase onset. On the other side of the anaphase switch, centromeres very probably function as targets for regulated proteolysis of the 'glue' that holds sister chromatids together.

Abstract Mitotic nuclear division is the process that ensures proper segregation of duplicated copies of the genome at cell division. The orderly segregation of genetic information depends on its reorganization into condensed chromosomes, and on the assembly and functioning of the mitotic spindle. The mitotic spindle is the machinery that segregates sister chromatids into the two daughter nuclei at mitosis. These events are precisely regulated to produce genetically identical daughter cells. In contrast, meiotic nuclear division is the process that generates genetic diversity in the offspring. Recombination of genetic information from cells with opposite mating types occurs at meiotic prophase. Meiosis encompasses two successive nuclear divisions: the first division involves cohesion of sister chromatids and disjunction of homologous chromosomes, while the second division disjoins sister chromatids in a process similar to mitotic di vision. In this chapter we first describe mechanisms of mitotic nuclear division with an emphasis on the mechanistic aspect of the mitotic spindle and chromosomes; next, we describe mechanisms of meiotic nuclear division with a particular emphasis on chromosome organization specific to meiosis.