Recognition of the centromere-binding protein Ndc10 by Scm3HJURP directs Cse4CENP-A deposition at native centromeres

Kinetochores connect centromeres to spindle microtubules, ensuring accurate chromosome segregation. Kinetochore assembly at centromeres depends on deposition of CENP-A, a histone H3 variant. Despite considerable progress, how CENP-A is specifically assembled on native centromeres remains an open question. Centromere-specific assembly of CENP-A occurs spontaneously in a cell-free extract system from the yeast Saccharomyces cerevisiae, but with low efficiency, suggesting that one or more factors are limiting. Here, we show Scm3HJURP, a specific chaperone for the centromeric histone Cse4CENP-A, is one such limiting component. The addition of a complex of Scm3, Cse4 and histone H4 strongly stimulates Cse4 assembly in a centromeric DNA sequence- and dose-dependent manner; in addition, improvements in both Cse4 and kinetochore assembly were more prominent in mitotic versus interphase (G1) extracts. Using the complemented cell-free reconstitution system, we show that distinct Scm3 interfaces with Cse4 and with the Ndc10 subunit of the yeast centromere DNA sequence-recognizing CBF3 complex are essential for centromere-specific Cse4 assembly and cell viability. Thus, recognition of Ndc10 directs Scm3-dependent and locus-specific centromeric histone deposition.