(A) Flow cytometry of wild type (WT) and mutant cells with the indicated genotypes that were arrested with α-factor and released into YPD medium in the presence or absence of 0.01% MMS. Cells were assayed every fifteen minutes. The rad9 rad24 and mad2 strains were tested twice, WT was tested six times, and rad9 rad24 mad2 was tested three times. (B) The percentage of large budded bi-nucleate cells (anaphase) from panel A for wild type and indicated mutant cells after release into medium with or without MMS. At least 100 cells were counted for each time point. Cell morphologies indicative of other phases of the cell cycle are in Figure S2. (C) Pds1-13 Myc stability of wild type and mutants cells. Endogenous Pds1 was tagged with 13 copies of the Myc epitope. Protein extracts from the cells in (A) were prepared and Western blot analysis was performed with 9E10 mouse anti-Myc monoclonal antibody. Upper half was in the absence of MMS and lower half was in the presence of MMS. Western blots with anti-Tub2 (tubulin) were for loading control. (D) Wild type and cells of the indicated genotypes were arrested with α-factor and released into YPD medium with or without 0.01% MMS. HU indicates wild type cells arrested with 0.1 M hydroxyurea. Samples were taken every hour and chromosomes were separated by CHEF.

(A) Flow cytometry of mec1-1 and rad9 rad24 tel1 cells arrested with α-factor and released to the cell cycle with or without MMS. (B) The percentage of cells that completed anaphase of isogenic sml1Δ cells (EKY490) and mutants of the indicated genotypes that were arrested with α-factor and had formed mating projections. The mean numbers of cells are graphed for cells with the indicated genotypes and the standard deviations are represented by error bars. Experiments were repeated independently three times and at least one hundred cells were counted for each time point in each experiment.

(A) Flow cytometry of mutant cells with the indicated genotypes that were arrested with α-factor and released into YPD medium with or without 0.01% MMS. Cells were assayed every fifteen minutes. The rad9 rad24 ndc10-1 cells were arrested with α-factor at 23°C for 3 hours, moved to 35°C for 1 hour to inactivate Ndc10, and then released to pre-warmed YPD at 35°C with or without MMS. All strains were tested twice except rad9 rad24 bub1 which was tested 3 times. (B) The percentage of large budded bi-nucleate cells from panel A for wild type and indicated mutant cells after released into medium without MMS (upper panel) or with MMS (lower panel). Cell morphologies indicative of other phases of the cell cycle are in Figure S4. (C) The number of rad9 rad24 bub3 cells that were budded with divided nuclei (anaphase) when grown in the presence or absence of MMS. Data from two independent experiments are represented. Solid lines are mean values and the dots are the independent measurements (range). (D) The SAC delay in response DNA damage requires APC^Cdc20. The number of CDC20-127 cells (upper panel) and CDC20-127 rad9 rad24 cells (lower panel) that were budded with divided nuclei (anaphase) when grown in the presence or absence of MMS. Data from three independent experiments are represented. The means are plotted and standard deviation is indicated by error bars. Analyses of morphologies indicative of other phases of cell cycle are in Figure S5D. (E) The SAC delay in response DNA damage requires Pds1. Number of cells that were budded with divided nuclei (anaphase) when grown in the presence of MMS are graphed. Closed circles are rad9 rad24 cells, open circles are rad9 rad24 mad2 cells, and triangles are rad9 rad24 pds1 cells. The arrows represent the time when 50% of the cells had completed anaphase when grown in the absence of MMS. Each point is the mean value of two independent experiments.

The lesion (indicated by the star) activates Mec1 and Tel1 which inhibits anaphase by phosphorylating Pds1 through the DNA damage checkpoint and independently inhibits Pds1 turnover by inhibiting APC^Cdc20 through the activity of the SAC.