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 division1. Despite their critical role, the structural organization of kinetochores assembled on centromeres with vastly distinct architectures across diverse species remains poorly understood2,3. 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.