Search

SummaryThe point centromere of budding yeast specifies assembly of the large multi-subunit kinetochore complex. By direct attachment to the mitotic spindle, kinetochores couple the forces of microtubule dynamics to power chromatid segregation at mitosis. Kinetochores share a conserved architecture comprising the centromere-associated inner kinetochore CCAN (constitutive centromere-associated network) complex and the microtubule-binding outer kinetochore KMN network. The budding yeast inner kinetochore additionally includes the centromere-binding CBF1 and CBF3 complexes. Here, we reconstituted the complete yeast inner kinetochore complex assembled onto the centromere-specific CENP-A nucleosome (CENP-ANuc) and determined its structure using cryo-EM. This revealed a central CENP-ANuc, wrapped by only one turn of DNA, and harboring extensively unwrapped DNA ends. These free DNA duplexes function as binding sites for two CCAN protomers, one of which entraps DNA topologically and is positioned precisely on the centromere by the sequence-specific DNA-binding complex CBF1. The CCAN protomers are connected through CBF3 to form an arch-like configuration, binding 150 bp of DNA. We also define a structural model for a CENP-ANuc-pathway to the outer kinetochore involving only CENP-QU. This study presents a framework for understanding the basis of complete inner kinetochore assembly onto a point centromere, and how it organizes the outer kinetochore for robust chromosome attachment to the mitotic spindle.

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an ∼171 bp α-satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while 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 which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, 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 α-satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces.One-Sentence SummaryThe human inner kinetochore CCAN complex tightly grips the linker DNA of the α-satellite CENP-A nucleosome.

Structural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, where they have key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through topological 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: proteobacterial MukBEF and eukaryotic cohesin. 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 favor 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.

Summary 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., Graphical abstract, Highlights • APC/C SUMOylation causes substantial rearrangement of the WHB domain of the APC2 subunit • The repositioned WHB domain prevents robust binding of the MCC to the APC/C • SUMOylation stimulates robust APC/C reactivation during anaphase onset, Yatskevich et al. show that SUMOylation of APC/C results in substantial structural rearrangements of the APC/C. These structural changes allow APC/C to be reactivated robustly upon anaphase entry and when spindle assembly checkpoint is silenced, ensuring that mitotic progression is not delayed.