RESUMEN
The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.
Asunto(s)
Proteínas de Ciclo Celular , Replicación del ADN , Inestabilidad Genómica , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Dominios Proteicos , Estabilidad Proteica , Mutación , Línea Celular , ADN HelicasasRESUMEN
Nuclear Cyclin D1 (Ccnd1) is a main regulator of cell cycle progression and cell proliferation. Interestingly, Ccnd1 moves to the cytoplasm at the onset of differentiation in neuronal precursors. However, cytoplasmic functions and targets of Ccnd1 in post-mitotic neurons are unknown. Here we identify the α4 subunit of gamma-aminobutyric acid (GABA) type A receptors (GABAARs) as an interactor and target of Ccnd1-Cdk4. Ccnd1 binds to an intracellular loop in α4 and, together with Cdk4, phosphorylates the α4 subunit at threonine 423 and serine 431. These modifications upregulate α4 surface levels, increasing the response of α4-containing GABAARs, measured in whole-cell patch-clamp recordings. In agreement with this role of Ccnd1-Cdk4 in neuronal signalling, inhibition of Cdk4 or expression of the non-phosphorylatable α4 decreases synaptic and extra-synaptic currents in the hippocampus of newborn rats. Moreover, according to α4 functions in synaptic pruning, CCND1 knockout mice display an altered pattern of dendritic spines that is rescued by the phosphomimetic α4. Overall, our findings molecularly link Ccnd1-Cdk4 to GABAARs activity in the central nervous system and highlight a novel role for this G1 cyclin in neuronal signalling.
Asunto(s)
Ciclina D1 , Quinasa 4 Dependiente de la Ciclina , Receptores de GABA-A , Animales , Ratones , Ratas , Ciclina D1/genética , Ácido gamma-Aminobutírico , Ratones Noqueados , Neuronas , Fosforilación , Receptores de GABA-A/genética , Quinasa 4 Dependiente de la Ciclina/genéticaRESUMEN
Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.
Asunto(s)
ARN Polimerasa I , Ubiquitina , Secuencia de Aminoácidos , ARN Polimerasa I/metabolismo , Proteómica , Proteínas de Ciclo Celular/metabolismo , ARN , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of RalB also inhibits cell growth in temozolomide-resistant GBM cells. Overall, our work shows that GBM cells are especially sensitive to Ral-GTPase availability. Therefore, we propose that the inactivation of Ral-GTPases may be a reliable therapeutic approach to prevent GBM progression and recurrence.
Asunto(s)
Glioblastoma , Animales , Proliferación Celular , Regulación hacia Abajo , GTP Fosfohidrolasas , Glioblastoma/genética , Humanos , RatonesRESUMEN
Post-translational modification by Small Ubiquitin-like Modifier (SUMO) proteins regulates numerous cellular processes. This modification involves the covalent and reversible attachment of SUMO to target proteins through an isopeptide bond, using a cascade of E1, E2 and E3 SUMOylation enzymes. Most functions of SUMO depend on the establishment of non-covalent protein-protein interactions between SUMOylated substrates and their binding partners. The vast majority of these interactions involve a conserved surface in the SUMO protein and a SUMO interacting motif (SIM), a short stretch of hydrophobic amino acids and an acidic region, in the interactor protein. Despite single SUMO-SIM interactions are relatively weak, they can have a huge impact at different levels, altering the activity, localization and stability of proteins, triggering the formation of macromolecular assemblies or inducing phase separation. Moreover, SUMO-SIM interactions are ubiquitous in most enzymes of the SUMO pathway, and play essential roles in SUMO conjugation and deconjugation. Here, we analyze the role of SUMO-SIM contacts in SUMO enzymes and targets and discuss how this humble interaction participates in SUMOylation reactions and mediates the outcome of this essential post-translational modification.
Asunto(s)
Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Ubiquitina , Ubiquitina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biomimética , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Daño del ADN , Procesamiento Proteico-Postraduccional , Proteostasis , Reparación del ADN por Recombinación , Proteínas Represoras , Ubiquitina , UbiquitinaciónRESUMEN
Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction.
Asunto(s)
Proteínas de Ciclo Celular/genética , Complejos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Fenómenos Biofísicos , Proteínas de Ciclo Celular/ultraestructura , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/ultraestructura , Proteínas de Unión al ADN/genética , Humanos , Interfase/genética , Mitosis/genética , Complejos Multiproteicos/ultraestructura , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/ultraestructura , Sumoilación/genética , CohesinasRESUMEN
The Smc5/6 complex plays essential roles in chromosome segregation and repair, by promoting disjunction of sister chromatids. The core of the complex is constituted by an heterodimer of Structural Maintenance of Chromosomes (SMC) proteins that use ATP hydrolysis to dynamically associate with and organize chromosomes. In addition, the Smc5/6 complex contains six non-SMC subunits. Remarkably, and differently to other SMC complexes, the Nse1 and Nse2 subunits contain RING-type domains typically found in E3 ligases, pointing to the capacity to regulate other proteins and complexes through ubiquitin-like modifiers. Nse2 codes for a C-terminal SP-RING domain with SUMO ligase activity, assisting Smc5/6 functions in chromosome segregation through sumoylation of several chromosome-associated proteins. Nse1 codes for a C-terminal NH-RING domain and, although it has been proposed to have ubiquitin ligase activity, no Smc5/6-dependent ubiquitylation target has been described to date. Here, we review the function of the two RING domains of the Smc5/6 complex in the broader context of SMC complexes as global chromosome organizers of the genome.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , Adenosina Trifosfato/química , Cromosomas/ultraestructura , ADN/química , Daño del ADN , Reparación del ADN , Humanos , Hidrólisis , Conformación Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Recombinación Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe , SumoilaciónRESUMEN
Replication of a damaged DNA template can threaten the integrity of the genome, requiring the use of various mechanisms to tolerate DNA lesions. The Smc5/6 complex, together with the Nse2/Mms21 SUMO ligase, plays essential roles in genome stability through undefined tasks at damaged replication forks. Various subunits within the Smc5/6 complex are substrates of Nse2, but we currently do not know the role of these modifications. Here we show that sumoylation of Smc5 is targeted to its coiled-coil domain, is upregulated by replication fork damage, and participates in bypass of DNA lesions. smc5-KR mutant cells display defects in formation of sister chromatid junctions and higher translesion synthesis. Also, we provide evidence indicating that Smc5 sumoylation modulates Mph1-dependent fork regression, acting synergistically with other pathways to promote chromosome disjunction. We propose that sumoylation of Smc5 enhances physical remodeling of damaged forks, avoiding the use of a more mutagenic tolerance pathway.
Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Sumoilación/genética , Cromátides/genética , Cromosomas/genética , ADN/genética , Daño del ADN/genética , Reparación del ADN/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6-Nse2 molecules engaged on DNA.
Asunto(s)
Proteínas de Ciclo Celular/química , ADN de Hongos/química , Complejos Multiproteicos/química , Proteína SUMO-1/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Ubiquitina-Proteína Ligasas/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Activación Enzimática , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Protein sumoylation is a reversible posttranslational modification that controls multiple processes during cell cycle progression. Frequently, SUMO synergistically targets various subunits in a protein complex to modulate its function, leading to what has been defined as protein group sumoylation. Different subunits in the RENT (regulator of nucleolar silencing and telophase) complex, including Net1, Sir2, and Cdc14, can be coupled to SUMO, making it difficult to ascertain the role of this modification. Here we describe a method to downregulate sumoylation in RENT, consisting in the fusion of a catalytic domain of the Ulp1 SUMO protease (Ulp Domain; UD) to the C-terminus of members in the complex using epitope tags as linkers. Targeting of the UD to specific loci can be simplified by transformation of PCR-amplified cassettes. The presence of the UD in the complex allows the concurrent downregulation of sumoylated species in the RENT complex, what can be easily monitored by pull-down of SUMO conjugates. This methodology can be applied to other protein complexes exhibiting group sumoylation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas de Ciclo Celular/genética , Clonación Molecular/métodos , Cisteína Endopeptidasas/genética , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Mutagénesis Sitio-Dirigida/métodos , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas Tirosina Fosfatasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Transformación GenéticaRESUMEN
The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, from end resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1-Top3-Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1's recombination functions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Cruciforme/metabolismo , RecQ Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Intercambio Genético , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Mutación , RecQ Helicasas/genética , Recombinación Genética/genética , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , SumoilaciónRESUMEN
Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis.
Asunto(s)
Ciclina D1/metabolismo , Citoplasma/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Paxillin/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Ciclina D1/deficiencia , Quinasa 4 Dependiente de la Ciclina/metabolismo , Regulación hacia Abajo/genética , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Ratas , Especificidad por Sustrato , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.
Asunto(s)
Anafase , Aurora Quinasas/metabolismo , Puntos de Control del Ciclo Celular , Replicación del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Actomiosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Anafase/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Hidroxiurea/farmacología , Viabilidad Microbiana/efectos de los fármacos , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
Modification of proteins by SUMO is essential for the maintenance of genome integrity. During DNA replication, the Mms21-branch of the SUMO pathway counteracts recombination intermediates at damaged replication forks, thus facilitating sister chromatid disjunction. The Mms21 SUMO ligase docks to the arm region of the Smc5 protein in the Smc5/6 complex; together, they cooperate during recombinational DNA repair. Yet how the activity of the SUMO ligase is controlled remains unknown. Here we show that the SUMO ligase and the chromosome disjunction functions of Mms21 depend on its docking to an intact and active Smc5/6 complex, indicating that the Smc5/6-Mms21 complex operates as a large SUMO ligase in vivo. In spite of the physical distance separating the E3 and the nucleotide-binding domains in Smc5/6, Mms21-dependent sumoylation requires binding of ATP to Smc5, a step that is part of the ligase mechanism that assists Ubc9 function. The communication is enabled by the presence of a conserved disruption in the coiled coil domain of Smc5, pointing to potential conformational changes for SUMO ligase activation. In accordance, scanning force microscopy of the Smc5-Mms21 heterodimer shows that the molecule is physically remodeled in an ATP-dependent manner. Our results demonstrate that the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair.
Asunto(s)
Proteínas de Ciclo Celular/genética , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Reparación del ADN por Recombinación , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfato/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Cromátides/ultraestructura , Daño del ADN , Replicación del ADN , ADN de Hongos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Sumoilación , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Cohesin is a protein complex that ties sister DNA molecules from the time of DNA replication until the metaphase to anaphase transition. Current models propose that the association of the Smc1, Smc3, and Scc1/Mcd1 subunits creates a ring-shaped structure that entraps the two sister DNAs. Cohesin is essential for correct chromosome segregation and recombinational repair. Its activity is therefore controlled by several posttranslational modifications, including acetylation, phosphorylation, sumoylation, and site-specific proteolysis. Here we show that cohesin sumoylation occurs at the time of cohesion establishment, after cohesin loading and ATP binding, and independently from Eco1-mediated cohesin acetylation. In order to test the functional relevance of cohesin sumoylation, we have developed a novel approach in budding yeast to deplete SUMO from all subunits in the cohesin complex, based on fusion of the Scc1 subunit to a SUMO peptidase Ulp domain (UD). Downregulation of cohesin sumoylation is lethal, and the Scc1-UD chimeras have a failure in sister chromatid cohesion. Strikingly, the unsumoylated cohesin rings are acetylated. Our findings indicate that SUMO is a novel molecular determinant for the establishment of sister chromatid cohesion, and we propose that SUMO is required for the entrapment of sister chromatids during the acetylation-mediated closure of the cohesin ring.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sumoilación , Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Daño del ADN , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologíaAsunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/análisis , Cromosomas/química , ADN de Cadena Simple/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análisisRESUMEN
Mitotic chromosome segregation requires the removal of physical connections between sister chromatids. In addition to cohesin and topological entrapments, sister chromatid separation can be prevented by the presence of chromosome junctions or ongoing DNA replication. We will collectively refer to them as DNA-mediated linkages. Although this type of structures has been documented in different DNA replication and repair mutants, there is no known essential mechanism ensuring their timely removal before mitosis. Here, we show that the dissolution of these connections is an active process that requires the Smc5/6 complex, together with Mms21, its associated SUMO-ligase. Failure to remove DNA-mediated linkages causes gross chromosome missegregation in anaphase. Moreover, we show that Smc5/6 is capable to dissolve them in metaphase-arrested cells, thus restoring chromosome resolution and segregation. We propose that Smc5/6 has an essential role in the removal of DNA-mediated linkages to prevent chromosome missegregation and aneuploidy.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromátides/metabolismo , Segregación Cromosómica , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas de Ciclo Celular/genética , Cromátides/química , Replicación del ADN/efectos de los fármacos , ADN de Hongos/química , ADN de Hongos/metabolismo , Genoma Fúngico/efectos de los fármacos , Metilmetanosulfonato/toxicidad , Mutación , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The structural maintenance of chromosome (SMC) proteins constitute the cores of three protein complexes involved in chromosome metabolism; cohesin, condensin and the Smc5-Smc6 complex. While the roles of cohesin and condensin in sister chromatid cohesion and chromosome condensation respectively have been described, the cellular function of Smc5-Smc6 is as yet not understood, consequently the less descriptive name. The complex is involved in a variety of DNA repair pathways. It contains activities reminiscent of those described for cohesin and condensin, as well as several DNA helicases and endonucleases. It is required for sister chromatid recombination, and smc5-smc6 mutants suffer from the accumulation of unscheduled recombination intermediates. The complex contains a SUMO-ligase and potentially an ubiquitin-ligase; thus Smc5-Smc6 might presently have a dull name, but it seems destined to be recognized as a key player in the maintenance of chromosome stability. In this review we summarize our present understanding of this enigmatic protein complex.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromosomas/fisiología , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/fisiología , Animales , Cromátides/fisiología , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/fisiología , Cromosomas/ultraestructura , Cromosomas Fúngicos/efectos de los fármacos , Cromosomas Fúngicos/fisiología , Cromosomas Fúngicos/efectos de la radiación , Cromosomas Fúngicos/ultraestructura , Reparación del ADN/fisiología , Replicación del ADN/fisiología , ADN de Hongos/genética , ADN Ribosómico/genética , Proteínas de Unión al ADN/fisiología , Humanos , Recombinación Genética/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiología , Schizosaccharomyces/citología , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/efectos de la radiación , Proteínas de Schizosaccharomyces pombe/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/genética , CohesinasRESUMEN
Translocations in chromosomes alter genetic information. Although the frequent translocations observed in many tumors suggest the altered genetic information by translocation could promote tumorigenesis, the mechanisms for how translocations are suppressed and produced are poorly understood. The smc6-9 mutation increased the translocation class gross chromosomal rearrangement (GCR). Translocations produced in the smc6-9 strain are unique because they are non-reciprocal and dependent on break-induced replication (BIR) and independent of non-homologous end joining. The high incidence of translocations near repetitive sequences such as delta sequences, ARS, tRNA genes, and telomeres in the smc6-9 strain indicates that Smc5-Smc6 suppresses translocations by reducing DNA damage at repetitive sequences. Synergistic enhancements of translocations in strains defective in DNA damage checkpoints by the smc6-9 mutation without affecting de novo telomere addition class GCR suggest that Smc5-Smc6 defines a new pathway to suppress GCR formation.