Figure 1.
CDC5 overexpression promotes adaptation by suppressing checkpoint signaling.
(A) Adaptation was measured by microcolony assay [33] in diploid strains carrying a combination of CDC5, cdc5-ad, or cdc5Δ alleles, or (B) in haploid strains with or without additional copies of integrated GAL-HA-CDC5. (C) Schematic model of checkpoint signaling. (D) Rad53 was analyzed by Western blots from cells that did or did not overexpress HA-CDC5 after DNA damage was induced by shifting to the non-permissive temperature of cdc13-1 strains or (E) by treating cells with 300 µg/ml zeocin. 2% galactose and 10 µg/ml nocodazole were added after 2 h of damage induction.
Figure 2.
CDC5 impinges on checkpoint signaling pathway at the step of Rad53 phosphorylation.
(A) After 2 h of 300 µg/ml zeocin treatment, 10 µg/ml nocodazole and 2% galactose were added to induce blank or HA-CDC5 for an additional 2 h. Cells were examined by fluorescence microscopy to visualize Ddc1-GFP or Ddc2-GFP localization. (B) Cells were DNA damaged by shifting cdc13-1 strains to 32°C for 2 h and then induced to express HA-CDC5. Rad9-FLAG was precipitated from lysates with Sigma α-FLAG congugated agarose beads. IP and lysates were analyzed by Western blotting with the indicated antibodies. (C) The reciprocal IP was performed as described in (B), immunoprecipitating Rad53 with the α-Rad53 (DAB001, from the Durocher lab) antibody on Protein A Dynabeads. Strains listed as+/−damage are cdc13-1 or CDC13, respectively.
Figure 3.
Suppression of Rad53 phosphorylation requires Cdc5 kinase activity but is independent of Ptc2, Ptc3, or Cdc14 phosphatases.
Rad53 phosphorylation was examined by Western blot from cells that have been damaged for 2 h before nocodazole and galactose were added to induce (A) CDC5, the kinase inactive cdc5-K110A, or adaptation defective cdc5-ad allele (B), or CDC5 in cells deleted for the PTC2 and PTC3 phosphatases. (C) Rad53 was immunoprecipitated with α-Rad53 from cells treated as above that express either HA-CDC5 or CDC14-Pk. In addition, rad9Δ and rad9Δ GAL-HA-CDC5 strains were examined as controls. Lysates and IP samples were analyzed by Western blotting with the indicated antibodies. (D) The experiment was performed as in parts (A) and (B) excepting that strains were transformed with a construct expressing a Ddc2-Rad53 fusion. Both WT Rad53 and the fusion protein were visualized with anti-Rad53 antibody.
Figure 4.
Rad53 interacts with and is phosphorylated by Cdc5.
(A) Rad53 was immunoprecipitated from strains shifted to 32°C for 2 h followed by galactose addition to induce the blank or HA-CDC5. As added controls, rad9Δ and CDC13 strains were also analyzed. Rad53 activity was measured by in situ autophosphorylation assay from the lysates. Asterisk denotes the lane with half the amount of sample as loaded in lane 4. (B) Lysates and IP samples from the experiment described in (A) were analyzed by Western blotting by the indicated antibodies. (C and D) In vitro kinase assays were performed with purified HA-Cdc5 kinase from (C) undamaged or (D) zeocin treated cells. The substrates were purified recombinant kinase-dead rad53 (D339A, listed as WT) in combination with R70A (FHA1) and/or R605A (FHA2) mutations.