FIG. 1.

Isolation of Msc1 as a multicopy suppressor of loss of checkpoint function. (A) Screening used to isolate the multicopy suppressors of loss of checkpoint function (see text for details). (B) chk1::ura cdc17-K42 strains transformed with either an empty vector or a plasmid with a genomic copy of msc1 (pmsc1) were grown to mid-log phase in liquid culture. Tenfold serial dilutions were made, and aliquots were spotted on plates. Plates were incubated at 25 or 32°C for 3 days. (C) Analysis of Chk1 phosphorylation in strains with defective DNA ligase activity of cdc17-K42 at 32°C. A cdc17-K42 strain with an integrated HA-tagged chk1 allele was transformed with either empty vector (lanes 1 and 2) or with pMsc1 (lanes 3 and 4). Strains were grown at 25°C to mid-log phase and then shifted to 32°C for 6 h. Protein was extracted by glass bead lysis, separated on an sodium dodecyl sulfate-polyacrylamide gel, transferred to nitrocellulose membrane, and blotted with antibody to the HA tag to detect the unphosphorylated and phosphorylated forms of Chk1. (D) A chk1::ura4 deletion strain was transformed with empty vector or plasmids containing genomic copies of msc1 or chk1. Strains were grown in liquid culture to mid-log phase, and 1,000 cells were plated and exposed to the indicated doses of UV light. All plates were incubated at 30°C for 3 days. The percentages of surviving colonies relative to those seen with unirradiated control plates were determined. Values shown are the averages of three independent experiments.