Figure 5.

At 25°C, Cds1 is required to maintain normal growth of polαts13 mutant and is activated. (A) Double mutant polαts13 cds1Δ has reduced growth rate at 25°C compared with the respective single mutant cds1Δ and polαts13. Serial dilutions of exponentially growing cells at 25°C by 10-fold were spotted on YES plates. Plates were incubated at 25°C for 3 d. (B) Double mutant polαts13 cds1Δ displays an elongated phenotype compared with the respective single mutant. Shown are phenotypes of single mutant polαts13 and double mutant polαts13 cds1Δ at 25°C. Bar, 3.5 μm. (C) Cds1 kinase is activated in polαts mutants at 25°C. Cds1 protein was immunoprecipitated from logarithmically growing wild-type cells, (wt), wild-type cells treated with 20 mM hydroxyurea [wt(HU)], polαts11 and polαts13, and cds1Δ cells. The immunoprecipitated Cds1 proteins were used to assay for kinase activity using MBP as the substrate as described in MATERIALS AND METHODS. Shown here is the phosphorylation of MBP by Cds1 kinase derived from different strains. (D) The histogram shows that Cds1 kinase activity is fourfold higher in polαts11 and polαts13 as compared with the wild-type polα⁺ integrant DB10 cells. In DB10 cells treated with hydroxyurea, the Cds1 kinase activity is 25-fold higher than in untreated cells. The Cds1 kinase activity from polαts13 is defined as the 100% maximum activity.