FIG. 5.

Crb2-BRCT₂ mutants have a defective IR checkpoint response. (A) The checkpoint arrest triggered by IR is abbreviated in crb2-S548A, crb2-K619M, and crb2-S548AK619M cells. The indicated mutants in a cdc25-22 background were synchronized at late G₂ phase by incubation at 35.5°C for 2.5 h. Following 180 Gy IR irradiation, cultures were returned to the permissive temperature of 25°C. Cell division (septation) was assessed by staining cells with Calcofluor. The data shown are representative of multiple experiments. (B) Chk1 phosphorylation is impaired in crb2-K619M and crb2-S548AK619M mutants. Exponentially growing asynchronous cultures were irradiated with 0, 30, and 120 Gy and harvested immediately following irradiation. (C) ImageJ quantification of the data shown in panel B. (D) Chk1 phosphorylation is ablated in crb2-T215AS548A cells. (E) ImageJ quantification of the data shown in panel D. Error bars represent the standard deviations of results from three independent experiments. The strains used for the experiments shown in this figure are SAS4904 (crb2⁺), LLD3628 (crb2Δ), SAS4905 (crb2-S548A), SAS4906 (crb2-K619M), SAS4917 (crb2-S548AK619M), SAS4909 (chk1-HA crb2Δ), SAS4914 (chk1-HA crb2⁺), SAS4910 (chk1-HA crb2-S548A), SAS4911 (chk1-HA crb2-K619M), SAS4912 (chk1-HA crb2-T215AS548A), SAS4913 (chk1-HA crb2-S548AK619M), and SAS4915 (chk1-HA crb2-T215A). WT, wild type.