Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway
Figure 7
Analysis of Sae2 protein in CDC5 overexpressing cells.
(A,B) YEP+raffinose nocodazole-arrested cell cultures of wild type JKM and isogenic GAL1::CDC5-MYC strain, expressing SAE2-3HA allele, were transferred to nocodazole-containing YEP + raffinose + galactose (time zero). Cells were collected at the indicated times and then subjected to chromatin immunoprecipitation (ChIP) as described in Figure 6. Representative ChIP time-course analysis of protein-DSB association is shown before (Inputs) and after protein immunoprecipitation (IP). (B) Western blot analysis of protein extracts. (C) Western blot analysis of protein extracts prepared 3 hrs after HO induction and treated with or without λ phosphatase before gel electrophoresis. (D) YEP-raffinose growing cells of wild type and of wild-type JKM MATa-inc and isogenic GAL1::CDC5-MYC strains, expressing SAE2-3HA allele, were split in two. One half was treated with nocodazole to block cells in G2. Galactose was then added to the cultures to induce overproduction of Cdc5. Cells were collected at the indicated times after galactose addition. (B–D) Sae2-HA protein was analyzed by western blots using 12CA5 antibody; Rad53 protein was analysed by monoclonal antibody Mab.EL7.