Figure 4. The Cenp-N DNA binding groove is required for stable CCAN – Cenp-A^Nuc interactions.

a, Surface of the Cenp-LN module showing the Cenp-N DNA binding groove engaging the unwrapped DNA, indicating the 13 mutated Arg and Lys residues of Cenp-N. Inset: overview of CCAN–Cenp-A^Nuc showing the Cenp-A L1 loop. b, Size exclusion chromatograms of various CCAN^ΔCenp-C–Cenp-A^Nuc complexes. Wild type CCAN^ΔCenp-C forms a complex with Cenp-A^Nuc, but mutating the Cenp-N DNA binding groove weakens CCAN – Cenp-A^Nuc interactions (Extended Data Fig. 8c, d). The binding of both CCAN^ΔCenp-C and CCAN^ΔCenp-C-Cenp-N^Mut to H3^N-Cenp-A^Nuc is severely disrupted, with little complex formed (Extended Data Fig. 8g, h). The positions of complexes are indicated by arrows. (CCAN^ΔC = CCAN^ΔCenp-C). This experiment was performed independently in triplicate with similar results. c, The DNA-binding groove functions in vivo. Wild type Cenp-N (CHL4^WT) rescues the growth defect of the chl4Δ cse4-R73A mutant strain at 37 °C, whereas the Cenp-N^Mut (chl4^Mut) does not. WT: wild type strain. This experiment was performed independently ten times with similar results. d, Western blot demonstrates that Cenp-N^WT and Cenp-N^Mut are expressed at equivalent levels in the chl4Δ cse4-R73A mutant strain. e, Loading control. Coomassie-blue stained gel shows dynein and acetyl-CoA carboxylase. Experiments in d and e were performed independently in triplicate times with similar results. f, Two views showing a representation of dimeric CCAN–Cenp-A^Nuc complex with the second CCAN protomer generated by the dyad symmetry of Cenp-A^Nuc. Sites of contact to the outer kinetochore (KT) (through Cenp-U and Cenp-T) are indicated. For gel source data see Supplementary Fig. 1.