Figure 7. Mutation of S1333 to aspartic acid causes modest defects in completing DNA synthesis following UV radiation and in maintenance of the G₂ checkpoint.

(A–B) The indicated wild type, S1333A, or S1333D-ATR expressing cell lines were treated as indicated below, fixed, stained with propidium iodide, and DNA content analyzed by flow cytometry. (A) Cells were treated with HU for 24 hrs, released into media containing nocodazole for 0, 4, or 10 hrs, and harvested, along with asynchronously growing cells. (B) Cells were treated with or without 50 J/m² UV and incubated with nocodazole for 16 hrs. The percentage of cells in S-phase was quantitated in three experiments and the mean and standard error are shown. (C) Cells were treated with or without 8 Gy IR and incubated with nocodazole for 16 hrs. The integrity of the G₂ checkpoint was analyzed by measuring mitotic cells using flow cytometry for DNA content and phospho-specific antibody to histone H3 S10. Error bars represent the standard error of three independent experiments. The difference between IR-treated A6 and D7 cells is statistically significant (one-way Anova, p = 0.0137).