Fig. 6: Phosphorylation of sites 460 and 471 depends on upstream CDK sites.

a, b Selection coefficients of the indicated mutants from the WT/E screen (a) or multiple screens (b). Error bars represent standard deviation, shown is an average of n = 3 biological replicates for all mutants except AAAAAAAA and EEEEEEEEE, which are an average of n = 6 biological replicates. Significance was tested using ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Asterisks indicate significantly different from WT (SC = 0), ****P < 0.0001, ***P < 0.0005, **P < 0.005, *P < 0.05, ns non-significant. Exact P values are included in the Source Data. c Transcriptional activation assay of fusion constructs containing the LexA-DBD fused to the Hcm1 TAD with the indicated phosphosite mutations, or WT or EV controls. Error bars represent standard deviations, shown is an average of n = 3 replicates. Significance was tested using repeated measures one-way ANOVA with Dunnett’s multiple comparisons test. Asterisks indicate significantly different from WT, ****P < 0.0001, ***P < 0.0005, **P < 0.005, *P < 0.05, ns non-significant. Exact P values are included in the Source Data. Supplementary Fig. 4b confirms that all fusion proteins are expressed at similar levels. The color gradient from blue (hcm1-8A) to red (hcm1-8E) corresponds to increasing numbers of phosphomimetic mutations (a–c). d Phos-tag western blot of the indicated 3V5-tagged Hcm1 proteins (top panel) and standard western blots showing expression of the indicated proteins (lower panels). PSTAIRE is shown as a loading control. Representative images from n = 3 biological replicates are shown. e Phos-tag western blots, as in (d) comparing Hcm1 mutants with A-P mutations in all S/T-P sites, except for the indicated sites in the TAD. The previously described 7N mutant²⁵ has the seven N-terminal S/T-P sites (all sites except those in the TAD) mutated to A-P, sites in the TAD have the indicated mutations. Representative images from n = 4 biological replicates are shown.