Fig. 4.

Both transcription and nuclear import defects lead to replication deficiency in cdc14. (A) Genes under control of Swi6p have partially decreased transcription in cdc14 mutants. Cells were arrested in G1 at 23 °C for 3 h, shifted at 37 °C for 30 min, and then released from G1 for 10 min. Expression levels were determined by qRT-PCR. All signals are normalized to TUB2 transcript, rDNA NTS1 - negative control. (B) Swi6p overexpression partially rescues the nucleolus segregation defect in cdc14. The cdc14 strain with Sik1p-RFP was transformed with either the pRS426gal vector (mock) or the pGAL:GST:SWI6 plasmid [derived from the library in (65)]. The cells were grown at 23 °C in plasmid-selective raffinose media, arrested with alpha factor in YPRaf for 2 h at 23 °C, galactose was added for 1 h/37 °C in the presence of alpha factor, and the cells were the released into YPRG/37 °C for 3 h. (C) RPA subunit imbalance in cdc14 mutants. Cells were arrested in G1 at 23 °C and released at 37 °C for 3 h untreated (37°), with hydroxyurea (HU, 0.2 M), with MMS (0.01%) or with nocodazole (NZ). Alpha-factor arrested cells were incubated at 37° for 1 h. Rfa2p detection: anti-GFP antibodies, Rfa1p detection: by specific antibodies. (D) Rfa2p phosphorylation is altered in cdc14 mutants even when expression is induced to wild-type level. Strains were arrested in G1 at 23 °C and then released to 37 °C or, in the presence of hydroxyurea (HU, 0.2 M), to 23 °C. Rfa2p detection as in E. The Mec1-dependent Rfa2p Ser-122 phosphorylation was detected with a specific antibody. Cdc28p levels (anti-PSTAIRE antibody): the loading control. (E) Rfa2p-GFP is largely delocalized from the nucleus in arrested cdc14 cells. Cultures were grown in YPD to log phase at 23 °C and shifted to 37 °C for 3 h. (F) Rfa1p and Rfa2p accumulate in cytoplasm in cdc14 mutants. Both cultures were arrested at 23 °C for 3 h, shifted at 37 °C for 30 min and released at 37 °C, samples were collected at indicated time points. Localization of Rfa1p and Rfa2p in nuclear (nuc) and cytoplasmic (cyt) fractions was analyzed by western blotting with specific polyclonal antibodies. Hmo1: chromatin and loading control. (G) Rfa2p does not interact with Kap60p-Kap95p importin complex in cdc14. Cells were arrested at 23 °C for 3 h, shifted at 37 °C for 30 min and split in half, with one half released from G1 at 37 °C. Rfa2p was immunoprecipitated from both G1 and anaphase cells, and analyzed with anti-Rfa2, anti-Kap60, and anti-Kap95 antibodies.