(A) The CENP-TCnn1 pathway is required for viability when the Mis12c assembly pathway is compromised. Dsn1-3A is synthetic lethal with cnn1Δ and deletions of other genes (MCM22 and CHL4) in the CENP-TCnn1 recruitment pathway. A dsn1-3A strain (SBY14170) was crossed to cnn1Δ (SBY13386), mcm22Δ (SBY6997), and chl4Δ (SBY8788). The meiotic products (tetrads) of the resulting diploids are oriented left to right, haploid spores were genotyped, and double mutants are indicated with circles. (B) A dsn1-3A mcm22-AID double mutant is lethal when treated with auxin. Serial dilutions of the following yeast strains were plated on the indicated media: WT (SBY3), dsn1-3A-3Flag (SBY14170), mcm22-3HA-AID OsTIR1 (SBY17982), and dsn1-3A-3Flag mcm22-3HA-AID OsTIR1 (SBY18171). (C) The CENP-TCnn1 pathway recruits Ndc80 when Mis12 complex assembly is compromised. Assembly was performed with extracts from HU-arrested strains that were treated with auxin: DSN1-3Flag OsTIR1 (SBY14131), DSN1-3Flag mcm22-3HA-AID OsTIR1 (SBY18044), dsn1-3A-3Flag OsTIR1 (SBY14169), and dsn1-3A-3Flag mcm22-3HA-AID OsTIR1 (SBY18034). Extracts in Figure 7—figure supplement 2. (D) WT (SBY18498) and dsn1-3A mcm22-3HA osTIR1 (SBY18324) cells containing MTW1-3GFP were released from G1 and kinetochores were analyzed by fluorescence microscopy during metaphase. The percentage of cells containing mono-lobed, bi-lobed or scattered kinetochores was quantified and a representative picture of the bi-lobed and scattered categories is shown above the graph. The p value for the difference between WT and the double mutant for bi-lobed kinetochores is 0.04 and for scattered kinetochores is 0.036. (E) The sequential order of kinetochore subcomplex recruitment to the DNA, as determined from our data and from (Pekgöz Altunkaya et al., 2016). Dotted lines indicate physical interactions.