Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. We now find that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, we show that the Mis12 complex component Nnf1 interacts directly with CENP-C in vitro. To test whether CENP-C's N-terminal part was sufficient to recruit KMN proteins, we targeted it to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis.

The multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential self-monitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells.

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4–DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1–233) homodimer at 2.3 Å resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.

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Aurora kinases A (also known as Aurora, Aurora-2, AIK, AIR-1, AIRK1, AYK1, BTAK, Eg2, MmIAK1 and STK15), Aurora B (also known as Aurora-1, AIM-1, AIK2, AIR-2, AIRK-2, ARK2, IAL-1 and STK12) and Aurora C (also known as AIK3) participate in several biological processes, including cytokinesis and dysregulated chromosome segregation. These important regulators of mitosis are over-expressed in diverse solid tumors. One member of this family of serine-threonine kinases, human Aurora A, has been proposed as a drugable target in pancreatic cancer. The recent determination of the three-dimensional structure of Aurora A has shown that Aurora kinases exhibit unique conformations around the activation loop region. This property has boosted the search and development of inhibitors of Aurora kinases, which might also function as novel antioncogenic agents.