RESUMEN
Structural maintenance of chromosomes (SMC) complexes are key organizers of chromosome architecture in all kingdoms of life. Despite seemingly divergent functions, such as chromosome segregation, chromosome maintenance, sister chromatid cohesion, and mitotic chromosome compaction, it appears that these complexes function via highly conserved mechanisms and that they represent a novel class of DNA translocases.
Asunto(s)
Cromátides , Cromosomas/metabolismo , ADN/química , ADN/metabolismo , Complejos Multiproteicos/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Cromosomas/química , Cromosomas/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Mitosis , Complejos Multiproteicos/química , Regiones Promotoras Genéticas , Recombinación V(D)J , CohesinasRESUMEN
Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.
Asunto(s)
Microscopía por Crioelectrón , Cinetocoros , Proteínas Asociadas a Microtúbulos , Modelos Moleculares , Proteínas Nucleares , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Fosforilación , Aurora Quinasa B/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Conformación Proteica , Proteínas Cromosómicas no HistonaRESUMEN
Microtubule (MT) filaments, composed of α/ß-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/ß-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/ß-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.
Asunto(s)
Microscopía por Crioelectrón , Microtúbulos , Modelos Moleculares , Saccharomyces cerevisiae , Huso Acromático , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Microtúbulos/química , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Tomografía con Microscopio Electrónico , Conformación Proteica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/ultraestructuraRESUMEN
Kinetochores are large protein complexes built on centromeric chromatin that mediate chromosome segregation. The inner kinetochore, or constitutive centromere-associated network (CCAN), assembles onto centromeres defined by centromere protein A (CENP-A) nucleosomes (CENP-ANuc), and acts as a platform for the regulated assembly of the microtubule-binding outer kinetochore. Recent cryo-EM work revealed structural conservation of CCAN, from the repeating human regional centromeres to the point centromere of budding yeast. Centromere recognition is determined mainly through engagement of duplex DNA proximal to the CENP-A nucleosome by a DNA-binding CENP-LN channel located at the core of CCAN. Additional DNA interactions formed by other CCAN modules create an enclosed DNA-binding chamber. This configuration explains how kinetochores maintain their tight grip on centromeric DNA to withstand the forces of chromosome segregation. Defining the higher-order architecture of complete kinetochore assemblies with implications for understanding the 3D organisation of regional centromeres and mechanisms of kinetochore dynamics, including how kinetochores sense and respond to tension, are important future directions.
Asunto(s)
Cromatina , Cinetocoros , Humanos , Cinetocoros/metabolismo , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Centrómero/metabolismo , Nucleosomas , ADN/metabolismoRESUMEN
The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo-electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-ANuc). This revealed a central CENP-ANuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-ANuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Cinetocoros/metabolismo , Microscopía por Crioelectrón , Proteína A Centromérica/genética , Saccharomycetales/genética , Subunidades de Proteína/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Centrómero/metabolismo , Saccharomyces cerevisiae/genética , ADN , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismoRESUMEN
Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-ANuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.
Asunto(s)
Proteína A Centromérica , Cinetocoros , Nucleosomas , Centrómero/química , Proteína A Centromérica/química , Microscopía por Crioelectrón , ADN/química , Humanos , Cinetocoros/química , Nucleosomas/química , Unión ProteicaRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that controls cell cycle transitions. Its regulation by the spindle assembly checkpoint (SAC) is coordinated with the attachment of sister chromatids to the mitotic spindle. APC/C SUMOylation on APC4 ensures timely anaphase onset and chromosome segregation. To understand the structural and functional consequences of APC/C SUMOylation, we reconstituted SUMOylated APC/C for electron cryo-microscopy and biochemical analyses. SUMOylation of the APC/C causes a substantial rearrangement of the WHB domain of APC/C's cullin subunit (APC2WHB). Although APC/CCdc20 SUMOylation results in a modest impact on normal APC/CCdc20 activity, repositioning APC2WHB reduces the affinity of APC/CCdc20 for the mitotic checkpoint complex (MCC), the effector of the SAC. This attenuates MCC-mediated suppression of APC/CCdc20 activity, allowing for more efficient ubiquitination of APC/CCdc20 substrates in the presence of the MCC. Thus, SUMOylation stimulates the reactivation of APC/CCdc20 when the SAC is silenced, contributing to timely anaphase onset.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Sumoilación , Ciclosoma-Complejo Promotor de la Anafase/química , Ciclosoma-Complejo Promotor de la Anafase/ultraestructura , Línea Celular Tumoral , Células HEK293 , Humanos , Mitosis , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, with key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through the entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: MukBEF from Escherichia coli, and cohesin from Saccharomyces cerevisiae. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head-kleisin module, situated at opposite ends of the arms. Our findings favour SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.