Regulation of chromosome bi-orientation and sister chromatid disjunction during cell division

Maintenance of a stable genome requires accurate DNA replication during S phase and error-free chromosome segregation during cell division. To allow proper chromosome segregation it is essential that the duplicated chromosomes (sister chromatids) remain linked in early mitosis and become attached to microtubules that emanate from opposite spindle poles, a process referred as chromosome bi-orientation. Time to accomplish bi-orientation is provided by the mitotic checkpoint, which senses the attachment status of kinetochores. Kinetochores are large multi-subunit protein complexes that assemble at the centromeric regions of chromosomes and mediate chromosome attachment to the mitotic spindle. The mitotic checkpoint prevents anaphase onset until all sister chromatids have established end-on attachments with microtubules of the mitotic spindle. The highly conserved Chromosomal Passenger Complex (CPC) (consisting of Aurora B, Inner CENtromere Protein (INCENP), survivin and borealin) is indispensable for chromosome bi-orientation and proper functioning of the mitotic checkpoint, and one of the aims of this thesis was to understand how this protein complex controls chromosome bi-orientation and faithful segregation of the sister chromatids. The enzymatic core of the complex, Aurora B kinase, is a member of the Aurora family of serine/threonine kinases, which includes Aurora A and Aurora C in addition to Aurora B. Aurora B requires the other CPC members for its activation (INCENP C-terminus) and localization (INCENP N-terminus that interacts with borealin and survivin), and they most likely also guide substrate specificity. In prometaphase and metaphase the CPC localizes at the inner centromeric region of the chromosomes (i.e. the region between the two sister-kinetochores) and this was crucial to its role in promoting chromosome bi-orientation. However this view was challenged by work in budding yeast from Campbell and Desai. In Chapter 2 we therefore investigated the importance of inner centromere localization of the CPC for chromosome bi-orientation in human cells. Furthermore, since the function of the CPC is dictated by the substrates that are phosphorylated by Aurora B, we developed a chemical genetic approach for this kinase to identify novel substrates of the CPC (Chapter 3). One of these substrates, Rif1, caught our attention because its localization appeared to overlap with Aurora B in anaphase. However, in the course of studying Rif1 we revealed a thus far unrecognized function for this protein in maintaining genomic integrity through the resolution of Ultrafine-Fine DNA Bridges (UFBs) in anaphase, independent of Aurora B (Chapter 5).