Fig. 2. Failure to undergo the DNA damage-induced mobility shift correlates with lack of nuclear localization. (A) Strains expressing the indicated catalytic domain mutants with three HA tags at the C-terminus were expressed from the chk1 promoter on the plasmid pSP1 in a chk1::ura4 disruption strain (NW158). Cells in mid-log phase were exposed to 20 µM CPT for 2 hours. Lysates were prepared, separated by SDS-PAGE and examined by western blot analysis for the HA tag on Chk1. Data is not shown for Chk1G108S, but it shows no evidence of a mobility shift in response to CPT treatment. (B) Strains expressing the indicated catalytic domain mutants with GFP fused to the C-terminus were expressed in a chk1::ura4 disruption strain (NW158) from the chk1 promoter on the plasmid pSP1. Cells were visualized with a fluorescence microscope. Chk1⁺ is wild-type Chk1-GFP. Alleles of Chk1 are indicated by the mutated amino acid number and substitution. (C) cDNAs encoding wild-type Chk1 or Chk1S345A fused to GFP were expressed from the nmt1 promoter on the pREP1 vector under repressing conditions in a chk1::ura4 disruption strain (NW158). Live cells were visualized by fluorescence microscopy.