The genetic interaction between DOA1 and CDC28 is ubiquitin dependent. (A) Ten-fold dilutions of wild-type, cdc28-as1 and doa1Δ single mutants and cdc28-as1 doa1Δ double mutants, either transformed with an empty vector (EV) or with a high-copy plasmid encoding UBI4, were spotted on YPD with DMSO or on YPD with increasing doses of 1-NM-PP1. (B) Overexpression of ubiquitin advances cell cycle entry in doa1Δ single mutants and restores the cell cycle entry defect of doa1Δ cdc28-as1 double mutants. (C) Log-phase cultures of wild-type cells, doa1Δ and cdc28-as1 single mutants were treated with 10 μM of 1-NM-PP1 for 10 min, after which 400 μM cycloheximide (CH) was added for the indicated times. Cell lysates were analyzed by Western blotting using anti-ubiquitin antibodies.

The DOA1–RAD6–BRE1 pathway promotes efficient cell cycle entry. (A) Log-phase cultures were synchronized with α-factor and released into YPD with nocodazole (to prevent cells from entering a second round of the cell cycle). At the indicated time points, samples were taken for analysis by flow cytometry. (B) Overexpression of ubiquitin does not ameliorate the cell cycle defect of rad6Δ single mutants and rad6Δ cdc28-as1 double mutants. Log-phase cultures of strains transformed with either empty vector (EV) or a high-copy vector harboring UBI4 were analyzed by flow cytometry as described in A. The sub-G1 peak is the result of altered cell morphology and is not due to sub-G1 DNA content.

Rad6 and Bre1 localize to cyclin genes to promote cyclin transcription. (A–D) Rad6 and Bre1 localize to cyclin genes. α-Factor–arrested strains expressing either RAD6–TAP or BRE1–TAP [both from the TAP tag collection (39)] were released into YPD for 30 and 60 min, fixed with formaldehyde, and processed for ChIP using TAP antibodies conjugated to protein A/G magnetic beads (“ChIP: αTAP”) or beads without antibody (“ChIP: no antibody”). Chromatin from a similarly treated, untagged wild-type strain served as negative control. ChIPs were analyzed by quantitative RT-PCR using primers specific for the transcription start sites of CLN2 (A), the ORF region of CLN2 (B), the transcription start site of CLB5 (C), the ORF region of CLB5 (D), and a transcriptionally inactive region of ChrV that contains no genes (E). The location of the amplified PCR products is indicated above the figures. TSS, transcription start site; ORF, open reading frame. (F) RAD6 and BRE1 are important for cyclin transcription. α-factor–arrested WT cells, rad6Δ, and bre1Δ mutants were released into YPD for 0, 20, 40, 60, or 80 min, and CLN2 and CLB5 mRNA levels were determined as described in Materials and Methods; CLN2 and CLB5 values were normalized to ACT1. (G) ACT1 expression does not depend on the Rad6–Bre1 pathway. ACT1 mRNA levels were determined as described above. (H) Deletion of WHI5 advances the timing and amplitude of CLN2 expression in rad6Δ mutants. CLN2 mRNA levels were determined as described above and normalized to CLN2 mRNA levels of the rad6Δ single mutant at 0 min.

The CGA screen identifies three classes of genes that genetically interact with CDC28. Network diagram summarizing genetic interactions with CDC28. Genes are colored according to their GO biological process (some were manually annotated from the literature).

Components of the RAD6-BRE1-LGE1 pathway genetically interact with CDC28. (A) Overexpression of ubiquitin does not restore growth of cdc28-as1 rad6Δ double mutants. Ten-fold dilutions of wild-type, cdc28-as1 and rad6Δ single mutants and cdc28-as1 rad6Δ double mutants, transformed with either an empty vector or a high-copy plasmid harboring UBI4, were spotted on YPD with DMSO or on YPD with 50 nM of 1-NM-PP1. (B) CDC28 genetically interacts with the RAD6–BRE1–LGE1 branch of the RAD6 pathway. Ten-fold dilutions of the indicated mutants were spotted on YPD with DMSO or on YPD with 1-NM-PP1.

The RAD6–BRE1–LGE1 pathway functions in parallel to WHI5 to mediate cell cycle entry. (A) α-Factor-arrested cultures of WT cells, cdc28-as1 and rad6Δ single mutants and rad6Δ cdc28-as1 double mutants were released into YPD supplemented with 15 μg/mL nocodazole (to prevent a second round of the cell cycle) for the indicated times and analyzed by flow cytometry. (B) Deletion of WHI5 suppresses the cell cycle defect of rad6Δ single and rad6Δ cdc28-as1 double mutants. Cell cycle entry was studied as in A.