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During cell division, interactions between microtubules and chromosomes are mediated by the kinetochore, a proteinaceous structure located at the primary constrictionof chromosomes. In addition to the centromere histone CENP-A, other members of the Constitutive Centromere Associated Network (CCAN), participate in the formation of a chromatin-associated scaffold that supports kinetochore structure. We performed a targeted screen analysing unfolded centrochromatin from centromere protein (CENP) depleted chromosomes. Our results revealed that CENP-C and CENP-S are critical for the stable folding of mitotic kinetochore chromatin. Multi‐peak fitting algorithms revealed the presence of an organised pattern of centrochromatin packing consistent with arrangement of CENP‐A containing nucleosomes into up to five chromatin “subunits” – each containing roughly 20‐30 nucleosomes. These subunits could be either layers of a boustrophedon or small loops of centromeric chromatin.
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