Fig. 4: S-CDK phosphorylation stimulates ATPase activity of Yta7.

a SDS-PAGE and Coomassie staining of purified S-CDK from nocodazole-treated cells. b In vitro kinase assay using purified S-CDK with both WT and mutant forms of Yta7 protein (WT, ATP-binding, Phospho) (Fig. 2b, Supplementary Fig. 2b). Incorporation of [³²P-γ]-ATP into Yta7 by S-CDK was visualized using autoradiography after separation by SDS-PAGE. Asterisk shows auto-phosphorylation of S-CDK. c Reaction scheme of the determination of the ATPase activity of Yta7. Purified Yta7 variants were pre-phosphorylated with S-CDK (omitted for controls) and treated with inhibitor Sic1 before re-purification. d ATP hydrolysis rates by re-purified Yta7 WT and Yta7 ATP-binding-mutants and Phospho-mutants in the presence of H3 acetylated chromatin was obtained by measuring the oxidation of NADH, which was used to regenerate hydrolyzed ATP. Error bars depict standard errors of three independent experiments. Mean values and standard deviations (S.D.) in d were obtained from three biological replicates. P-values were obtained by using two-tailed unpaired t-test calculations. N.S. means statistically non-significant.