During mitosis, the inner layer of the kinetochore, the 16-subunit Constitutive Centromere Associated Network (CCAN), recognizes the centromere and recruits the outer kinetochore to physically link the sister chromatids to the spindle microtubules. The CCAN subunits were originally identified as proteins that specifically copurify with centromeric nucleosomes containing the histone H3 variant CENP-A. The purifications also included the histone chaperone FAcilitates Chromatin Transcription (FACT), a heterodimer of SPT16 and SSRP1. FACT promotes the assembly and disassembly of nucleosomes during transcription, replication and DNA repair and is essential for maintaining histone variants and posttranslationally modified histones on chromatin. At the centromere, FACT has been implicated in replenishing CENP-A levels in other species and interacts with the histone fold domain of CENP-T/-W subunits of CCAN in humans.

In this study, I provide an in-depth characterization of the FACT/CCAN complex using biochemical reconstitution. I demonstrated that FACT directly binds to CCAN subcomplexes CENP-C and CENP-OPQUR in addition to CENP-TW, forming a stable 18-subunit complex of equimolar stoichiometry. This interaction requires phosphorylation of FACT by the constitutively active casein kinase 2. The interaction of CCAN with DNA results in the release of FACT from the complex, suggesting that FACT binds a pool of CCAN that is not stably incorporated into chromatin. In the context of recent cell biological data, I propose that FACT stabilizes CENP-TW and potentially the entire CCAN at the centromere. This may be especially relevant during transcription, when proteins need to be transiently removed from the DNA.

Furthermore, I obtained a low-resolution 3D reconstruction of the CENP-A loading machinery determined by cryo-EM, revealing that the Mis18 core complex assembles in an elongated hexamer. The Yippee domains adopt a linear α-β-α-α-β-α configuration, while the C-terminal helical bundles extend toward the same direction, tilted relative to each other. Structural prediction by AlphaFold2 illustrated the binding of M18BP1 and HJURP to the Mis18 core complex in strong agreement with the previous biochemical characterization.