Cryo-EM structure of the complete inner kinetochore of the budding yeast point centromere

Abstract

The point centromere of budding yeast specifies assembly of the large kinetochore complex to mediate chromatid segregation. Kinetochores comprise the centromere-associated inner kinetochore (CCAN) complex and the microtubule-binding outer kinetochore KNL1-MIS12-NDC80 (KMN) network. The budding yeast inner kinetochore also contains the DNA binding centromere-binding factor 1 (CBF1) and CBF3 complexes. We determined the cryo–electron microscopy structure of the yeast inner kinetochore assembled onto the centromere-specific centromere protein A nucleosomes (CENP-A Nuc ). This revealed a central CENP-A Nuc with extensively unwrapped DNA ends. These free DNA duplexes bind two CCAN protomers, one of which entraps DNA topologically, positioned on the centromere DNA element I (CDEI) motif by CBF1. The two CCAN protomers are linked through CBF3 forming an arch-like configuration. With a structural mechanism for how CENP-A Nuc can also be linked to KMN involving only CENP-QU, we present a model for inner kinetochore assembly onto a point centromere and how it organizes the outer kinetochore for chromosome attachment to the mitotic spindle.

Recognition of centromere‐specific histone Cse4 by the inner kinetochore Okp1‐Ame1 complex

“…Therefore, Cse4 can bind the assembled COMA-Nkp1-Nkp2 complex. Because Nkp1-Nkp2 do not displace Cse4 END from Okp1-Ame1, their inclusion cannot explain the inability of Cse4 END to bind the reconstituted Ctf19c in biochemical experiments (Dendooven et al, 2023).…”

“…Because the Okp1-Ame1 head domain is shifted in the crystal structure as described above, it is not clear from these comparisons whether Nkp1-Nkp2 would need to partly dissociate from Okp1-Ame1 to accommodate Cse4 END (Fig EV1F). Indeed, a recent cryo-EM structure of COMA-Nkp1-Nkp2 shows partial Nkp1-Nkp2 dissociation from Okp1-Ame1 is possible (Dendooven et al, 2023). We used pulldown assays to test whether Nkp1-Nkp2 and Cse4 END compete for Okp1-Ame1 binding.…”

“…Molecular recognition of Cse4 by the Okp1-Ame1 heterodimer has not been resolved, despite recent cryo-EM structures that show in detail nearly all protein contacts that contribute to inner kinetochore assembly in yeast (Yan et al, 2018(Yan et al, , 2019Hinshaw & Harrison, 2019Guan et al, 2021;Dendooven et al, 2023). An experimental solution will constrain models for centromeric nucleosome binding by the Ctf19c.…”

Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules

“…4, D and E). The inner kinetochore generates a total of eight Ndc80c linkages to the microtubule ( 49 ). These Ndc80c molecules are readily accommodated by the 16 binding sites of Dam1c Ring , which is recruited through Ndc80c in a microtubule-dependent fashion ( 50 ).…”

“…( D ) Structural model of the complete yeast holo-kinetochore bound to a microtubule. Shown are the microtubule, Dam1c Ring , four KMN network complexes, two Ndc80 complexes, and the CCAN inner kinetochore complex (composed of two CCAN promoters, CBF1, CBF3, and CENP-A nucleosome) ( 49 ). The flexible linkers connecting CCAN to the KMN network and Ndc80c are not shown.…”