FIG. 6.

The rfc4-2 mutant is defective in the DNA damage checkpoint. (A) Exponentially growing cells of the indicated genotype were incubated with or without 0.1% MMS for 1 h. Both MMS-treated (lanes 4, 5, and 6) and -untreated (lanes 1, 2, and 3) cells were analyzed for Rad53 phosphorylation by Western blotting. (B) Exponentially growing wild-type and rfc4-2 cells were treated with or without UV light (60 J/m²), were incubated for 30 min, and then were analyzed for Rad53 phosphorylation by Western blotting (lanes 1 and 2). In addition, wild-type and rfc4-2 cells were synchronized in G₁ or G₂ with α-factor or nocodazole, respectively, and were treated and analyzed as above (lanes 3 to 6). Asterisks (∗) denote nonspecific bands. (C) The rfc4-2 mutant was tested for G₂/M arrest in response to lesions caused by cdc13. Four cdc13 cdc15 strains, DLY408 (WT), DLY409 (rad9), HSY1202 (rfc4-2), and HSY1204 (rfa1-t11), were synchronized in G₁ with α-factor at 23°C. Cells were then released from the G₁ block, shifted to 36°C, and incubated for 3.5 h. Cells were fixed and stained, and their morphology was analyzed by fluorescence microscopy. The percentage of cells arrested at G₂/M (large budded cell with a single nucleus at the neck) or at the cdc15 arrest point (large budded cell with two nuclei) is shown in the table in the lower right panel. Cells with nuclear DNA stretched between the mother and daughter compartments are not represented in the table and account for the percentages summing to less than 100.