A) cdc13-1 cells were synchronized in α-factor and released at 32°C. Cells were collected at indicated timepoints and lysed in TCA. For the YPE sample, WT cells were grown in YP media containing ethanol overnight and the cells were lysed in TCA. Proteins were separated on a 10% polyacrylamide SDS gel. Western analysis was used to detect the R subunit using an anti-Bcy1 (R subunit) antibody. For loading control see Figure S3A. B) cdc13-1 cells were grown at 22°C and the temperature was raised to 32°C for 120 min., cells were collected and TCA precipitated protein extracts were prepared. After re-solubilizing the TCA precipitated protein by boiling, the R subunit was isolated by immunoprecipitation and treated with alkaline phosphatase with or without phosphatase inhibitors as indicated. Separation and detection of the R subunit was carried out as described above. C) WT and cdc13-1 cells were grown at 22°C and raised to 32°C for 2 hours. 0.1% MMS, 10 µg/ml nocodazole were added to WT as indicated for the 2 hours the cells were at 30°C. Cells were lysed and proteins detected as in (A). Loading control Figure S3B. D) cdc13-1 and cdc13-1 mec1Δ cells were grown and treated as in (B), and detection of R subunit was carried out as in (A). Loading control Figure S3C, replicate experiments Figure S4.

A) Phosphorylation of the R subunit in response to DNA damage is dependent on Mck1. cdc13-1 MCK1 (WT), cdc13-1 mck1Δ and cdc13-1 mec1Δ cells were raised to 32°C for 120 min. Nocodazole was added to MCK1 cells for 120 min. Cells were lysed, and the R subunit was detected as described in Figure 1. Loading control for (A) in Figure S5A. Replicate of experiment in (A) is shown in Figure S5B. B) cdc13-1, cdc13-1 chk1Δ, cdc13-1 mck1Δ, cdc13-1 chk1Δ mck1Δ, and cdc13-1 chk1Δ tpk2Δ, cells were arrested in S-phase by addition of HU (at 22°C), the temperature was raised for 60 min and the cells were released into the cell cycle at 32°C. Cells were collected at the indicated time points, fixed in ethanol, and stained with DAPI to visualize the DNA and nuclear morphology. The number of cells that exhibited a late mitotic phenotype were counted. These strains were part of experiment in Figure S1. C) cdc13-1 tpk2Δ, cdc13-1 chk1Δ tpk2Δ (same as in Figure 5C) and cdc13-1 chk1Δ tpk2Δ mck1Δ cells were arrested in G1 with α-factor and released into the cell cycle at 32°C. Samples were taken at indicated times and analyzed as in (B). D) cdc13-1 and cdc13-1 mec1-21 cells transformed with either the high-copy vector pRS425 or MCK1 in pRS425 (OP MCK1) were grown at 22°C and arrested in G1 by addition of α-factor in YPD pH 3.9. The temperature was raised to 32°C and the cells were released from G1 into the cell cycle at 32°C in YPD pH 3.9. Cells were collected at the indicated time points and analyzed as in (B). Replicate experiments for (D–F) are shown in Figure S6A–S6D. E) Cells from (D) were monitored for the appearance of small buds. F) Cells from (D) were scored for the time it took to reach large budded state with an undivided nucleus. G) cdc13-1 (WT), cdc13-1 chk1Δ, cdc13-1 swe1Δ, cdc13-1 chk1Δ swe1, cdc13-1 chk1Δ mck1Δ cells were grown, arrested, and released as in (D).

A) TOP: R subunit (Bcy1) showing position of Cluster I and II on the N-terminus and the serines changed to alanines (asterisks) in the Cluster I and Cluster II mutants. BOTTOM: cdc13-1 bcy1Δ+BCY1, cdc13-1 bcy1Δ+bcy1CI, and cdc13-1 bcy1Δ+bcy1CII cells were grown at 22°C and the temperature was raised to 32°C for 120 min. Cells were collected, lysed and the proteins were analyzed as in Figure 1A. B) All strains are in cdc-13-1 bcy1Δ background: cdc13-1 bcy1Δ+BCY1 (WT), cdc13-1 bcy1Δ+bcy1CI (CI), cdc13-1 bcy1Δ+bcy1CII (CII), cdc13-1 chk1Δ bcy1Δ+CY1, cdc13-1 chk1Δ bcy1Δ+bcy1CI, and cdc13-1 chk1Δ bcy1Δ+bcy1CII cells were arrested in S-phase by addition of HU to the media. The temperature was raised to 32°C for 60 min and the cells were released into the cell cycle at 32°C. Aliquots were taken at the indicated times and each sample was analyzed as in Figure 2B. C) cdc13-1 bcy1Δ+BCY1, cdc13-1 chk1Δ bcy1Δ+BCY1, cdc13-1 bcy1Δ chk1Δ mck1Δ+BCY1, cdc13-1 bcy1Δ chk1Δ mck1Δ+bcy1CI, and cdc13-1 bcy1Δ chk1Δ mck1Δ+bcy1CII cells were analyzed as in Figure 2B. D) WT BCY1-GFP cells and cdc13-1 BCY1-GFP cells were grown at 22°C arrested in G1 with α-factor and released into the cell cycle at 32°C in the presence or absence of 10 µg/ml nocodazole. Localization of Bcy1-GFP in WT BCY1-GFP cells in late S/G2 was scored 50 min after release when the majority of cells were large budded. Bcy1-GFP localization was scored in the remainder of the cells 120 min. after release. To visualize GFP expression, cells were spotted on slides pre-coated with polylysine (Poly-L-lysine) and allowed to dry. GFP expression was analyzed using a Zeiss LSM 510 Meta confocal microscope. Graph represents average of three experiments. WT = Construct expressing BCY1, CI = Construct expressing bcy1CI, CII = Construct expressing bcy1CII. Replicate experiment shown in Figure S2.

A) cdc13-1, cdc13-1 chk1Δ, cdc13-1 zds2Δ, cdc13-1 chk1Δ zds2Δ, and cdc13-1 chk1Δ mck1Δ cells were arrested in S-phase using HU and released into the cell cycle at 32°C as in Figure 2B. Aliquots from each culture were taken at indicated times following release into the cell cycle, fixed and analyzed as in Figure 2B. Replicate experiment is shown in Figure S1B. B) TCA precipitated proteins from WT cells treated with nocodazole for 120 minutes, and TCA precipitated proteins from cdc13-1, cdc13-1 zds1Δ, and cdc13-1 zds2Δ cells grown at 32°C for 120 minutes and proteins were analyzed as in Figure 1A.

A) cdc13-1, cdc13-1 chk1Δ, cdc13-1 rad9Δ, cdc13-1 hxk2Δ, and cdc13-1 chk1Δ hxk2Δ cells were arrested in G1 using α-factor and released into the cell cycle at 32°C. Aliquots were removed at indicated time points and analyzed as in Figure 2B. B) cdc13-1, cdc13-1 chk1Δ, cdc13-1 cdc35-1, cdc13-1 cdc35-1 chk1Δ, cdc13-1 chk1Δ hxk2Δ cells were arrested in G1 with α-factor and released into the cell cycle at 22°C. 30 minutes following release from G1 the temperature was gradually raised so that 45 minutes after release from G1 the temperature was 35°C. Aliquots of the cells were taken at the indicated times and were analyzed as in Figure 2B. C) cdc13-1 tpk2Δ, cdc13-1 chk1Δ tpk2Δ, and cdc13-1 chk1Δ tpk2Δ hxk2Δ cells were released from a G1 block into the cell cycle at 32°C. Aliquots of the cells were taken at indicated timepoints and analyzed as in Figure 2B. The chk1Δ tpk2Δ hxk2Δ cells were run along with strains in Figure 2C, therefore the control strains are the same as those shown in Figure 2C. D) cdc13-1, cdc13-1 hxk2Δ cells were grown at 22°C and the temperature was increased to 32°C for 120 minutes. The cells were lysed and the proteins analyzed as in Figure 1A.

A) WT BCY1-GFP cells, cdc13-1 BCY1-GFP, cdc13-1 mck1Δ BCY1-GFP, cdc13-1 hxk2Δ BCY1-GFP, cdc13-1 zds1Δ BCY1-GFP and cdc13-1zds2Δ BCY1-GFP cells were grown at 22°C arrested in G1 with α-factor and released into the cell cycle at 32°C. Cells were observed under the microscope until large budded. Localization of Bcy1-GFP in WT BCY1-GFP cells in late S/G2 was scored 50 min after release when the majority of cells were large budded. Bcy1-GFP localization was scored in the remainder of the cells 120 min. after release. To visualize GFP expression, cells were spotted on slides pre-coated with polylysine (Poly-L-lysine) and allowed to dry. GFP expression was analyzed using a Zeiss LSM 510 Meta confocal microscope. Graph represents average of three experiments. Asterisks represent significance compared to cdc13-1 GFP-BCY1. * = p<0.05, ** = p<0.01, *** = p<0.001, NS = Not significant. All T-tests are two-tailed. B) Model, see text for details.