Figure 7. Dysfunctional Cik1/Kar3 leads to decreased DASH–kinetochore interaction.
A. Overexpression of CIK1-CC is lethal to ask1 mutants. Saturated cell cultures with indicated genotypes were 10-fold diluted and then spotted onto dropout plates containing glucose or galactose. B. cik1Δ mutant cells show decreased centromere association of Ask1. G1-arrested cdc13-1 ASK1-9myc and cdc13-1 cik1Δ ASK1-9myc were released to YPD medium at 34°C. The cells were collected at time 0 and 90 min for ChIP assay with anti-myc antibody. The PCR products with primers specific for CEN1, CEN3, and ACT1 are shown in the left panel and the quantified data are shown in the right. The ratio of the PCR yield (IP/input) in WT cells was standardized as one. The experiment was repeated three times. C. cik1Δ mutant cells exhibit normal association of Nnf1 with centromeric DNA. cdc13-1 NNF1-13myc and cdc13-1 cik1Δ NNF1-13myc cells were treated as in B. The PCR products with primers specific for CEN1, CEN3 and ACT1 are shown in the left panel and the quantified data are shown in the right. The experiment was repeated for three times.