Moreover, the treatment of fixed chromosomes with Lambda phosphatase did not lead to the detection of CENP-A between sister kinetochores with anti-CENP-A antibodies (Supplementary Fig.?1G). A-dependent phosphorylation of CENP-A at the inner centromere protects chromosomes against tension-induced cohesion fatigue until the last kinetochore is attached to spindle microtubules. Introduction The centromere is a specialized domain of the chromosome required for the faithful segregation of sister chromatids during mitosis. In higher eukaryotes, centromere identity is dependent on the incorporation of specific nucleosomes containing CENP-A, a centromere-specific variant of histone H31. However, centromere chromatin does not consist exclusively of CENP-A nucleosomes2C4, as canonical histone H3 nucleosomes are also present at the centromere. In all models of centromere chromatin structure in mitotic chromosomes, the CENP-A nucleosomes are found exclusively on the external face of the centromere, whereas the inner centromere contains exclusively H3 nucleosomes2, 4C7. The N-terminal tail of CENP-A differs considerably from that of H3, and between Cevimeline (AF-102B) species. This domain has been reported to be necessary for recruiting kinetochore components and for accurate chromosome segregation8, Cevimeline (AF-102B) 9. The N-terminal domain of histones is subject to numerous posttranslational modifications (PTMs) that influence all chromatin-related processes and PTMs of CENP-A have also recently been described10C12. It has been suggested that p-CENP-AS7 is involved in the maintenance of an active kinetochore during mitosis8, in chromosome alignment and correct chromosome segregation9, and in the completion of cytokinesis13. CENP-AS7 is partially phosphorylated by Aurora A during the prophase of mitosis9, and this phosphorylation is required for Chromosomal Passenger Complex (CPC) recruitment at centromeres during prometaphase and for the completion of CENP-AS7 phosphorylation by the Aurora B kinase9, 13C15. Both the Aurora kinases are involved in various processes required for accurate cell division. Aurora A localizes to spindle poles, where it is involved in centrosome maturation and separation16. Aurora B is recruited to the inner side of centromeres, between sister kinetochores, where it supervises chromosome biorientation by correcting erroneous microtubule/kinetochore attachments17. One of the major functions of the inner centromere is protecting the attachment between sister chromatids until the spindle assembly checkpoint (SAC) is satisfied. Sister chromatid cohesion must persist at the centromeres during the early stages of mitosis for correct chromosome biorientation and the establishment of tension between sister centromeres. Sister chromatid cohesion is mediated by the cohesin complex18. In vertebrates, the phosphorylation of the cohesin complex components by CDK1, PLK1 and Aurora B leads to the removal of most cohesins from the chromosome arms during prophase19C21. Until the onset of anaphase, cohesion at centromeres is protected by Shugoshin1 (Sgo1)22, 23. Bub1-mediated phosphorylation of CD350 the threonine 120 residue of histone H2A (H2AT120) at the centromeres is essential for Sgo1 recruitment during mitosis24. However, other mechanisms, including interaction with H3K9Me3-associated heterochromatin protein 1 and direct binding to the cohesin complex, are required for the targeting of Sgo1 to the centromere23, 25, 26. Moreover, in human cells, Sgo1 undergoes tension-dependent relocation from the inner centromere to the kinetochores27, 28. Sgo1 acts as a sensor of tension between sister kinetochores and promotes chromosome biorientation29, 30. However, despite the protective function of Sgo1, sustained tension between sister centromeres ultimately leads to progressive loss of sister chromatid cohesion. This stochastic and unprogrammed phenomenon is known as cohesion fatigue31, 32. We report here that, unlike the non-phosphorylated form of CENP-A, p-CENP-AS7 localizes to the inner side of the centromere during mitosis. The prevention of CENP-AS7 Cevimeline (AF-102B) phosphorylation or the depletion of Aurora A increases the number of cells displaying premature sister chromatid separation (PSCS). We show that, in addition to its known spindle pole localization, Aurora A is associated with centromeres during mitosis. We found that, besides its known role in Sgo1 targeting to centromeres, the Bub-1 kinase is required for recruiting Aurora A to centromeres and, thus, for CENP-AS7 phosphorylation. The loss.