Architecture and Function of Holocentric CENP-A-Independent Kinetochores

Kinetochores are essential macromolecular complexes that anchor chromosomes to the mitotic spindle to ensure faithful cell division ¹ . Despite their critical role, the structural organization of kinetochores assembled on centromeres with vastly distinct architectures across diverse species remains poorly understood ^2,3 . To address this question, we determined the cryo-EM structures of the inner kinetochore (CCAN) from the silkmoth Bombyx mori , an insect that lacks the canonical centromere-specifying histone variant CENP-A and exhibits chromosome-wide centromeric activity (holocentric). Our analysis reveals that B. mori CCAN assembles via atypical histone-fold protein dimerization into a self-contained, head-to-head dimer that topologically entraps and loops DNA, creating a point-centromere-like architecture. This structure also incorporates four previously uncharacterized Centromeric Subunit proteins that are evolutionarily repurposed from the outer kinetochore Dam1/DASH complex. Our work establishes this self-contained CCAN dimer as a key structural unit that forms the basis of a holocentric organization and suggests that large-scale centromere architectures can emerge from the modular arrangement of such discrete kinetochore units.