Fig. 4. Mutation of serine 345 to alanine or aspartic acid compromises checkpoint function and viability following exposure to DNA damage. (A) NW228 (chk1⁺), NW107 (chk1::ura4), NW442 (chk1S345A) and NW477 (chk1S345D) cells harboring the cdc25-22 temperature-sensitive mutant were grown to mid-log phase at 25°C, plated on agar plates, shifted to 36.5°C for 2 hours, treated with UV light, shifted back to 25°C, and monitored for entry into mitosis. Symbols for strains are shown; closed symbols, + UV; open symbols, - UV. (B) NW223 (chk1⁺), NW158 (chk1::ura4), NW444 (chk1S345A) and NW463 (chk1S345D) cells were grown to mid-log phase, spread on agar plates, exposed to UV, and incubated for approximately 3 days. Colony number was determined and is expressed as a percentage of colonies appearing on untreated plates. Symbols for strains are as in A. (C) NW227 (chk1⁺), NW157 (chk1::ura4), NW443 (chk1S345A) and NW479 (chk1S345D) cells harboring the cdc17-K42 temperature-sensitive mutant were grown to mid-log phase, resuspended at 1x10⁷ cells/ml, spotted onto agar plates at 10x dilutions, and incubated for approximately 3 days at 25°C, 32°C or 36°C. (D) Phosphorylation of Chk1 occurs in strains with the wee phenotype, which are dependent on chk1 for survival. Strains NW223 (wild-type), NW252 (mik1::ura4), NW253 (mik1::ura4 wee1-50), NW230 (wee1-50) and NW239 (rad1-1 wee1-50) were grown at 25°C and lysed for analysis by immunoblot with 12CA5 anti-HA antibody.

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