Measuring checkpoint arrest in a 1-DSB and 2-DSB strains

(A) Morphological categories of budding yeast cells using brightfield microscopy and DAPI staining were used to determine G2/M arrest. Cells that arrest at G2/M shift towards a large bud state. G2/M arrested cells that progress into anaphase. (B) Adaptation assay with 1 DSB strain on a YEP-Gal plate. G2/M arrest was determined based on cell morphology as shown in Fig. 1A. Data are shown from 3 independent experiments, error bars represent standard error of the mean (SEM). (C) Rad53 phosphorylation kinetics in 1 DSB strain by western blotting. Samples collected after the induction of DNA damage during the time course experiment and blotted with α-Rad53 to monitor DDC signaling. Α-Rad53 can both detect unphosphorylated and hyperphosphorylated Rad53 species. TIR1-myc was detected with α-Myc and serves as a loading control. (D) Profile of DAPI stained cells in a 1-DSB strain after DNA damage induction in liquid culture. Cells were grouped based on cell morphology and DAPI staining profiles, as explained below the graphs. (E) Same as (B) for a 2-DSB strain. (F) Same as (C) with a 2-DSB strain. (G) Same as (D) with a 2-DSB strain.

Checkpoint maintenance requires Ddc2, Rad9, Rad24, and Rad53 activity.

(A) Above, percentage of G2/M arrested cells in a 2-DSB Ddc2-AID strain after DNA damage induction in a liquid culture. Cultures were split 4 h after galactose treatment to induce DNA damage by GAL::HO and treated either with auxin (+IAA) (1mM) or with ethanol (Ctrl). Data are shown from 3 independent experiments with error bars representing standard error of the mean (SEM). The asterisk marks the time point when the percentage of large-budded G2/M cells returned to pre-damage levels. Below, western blots ran with samples collected at various time points during the same time course, probed with α-Rad53 to determine DDC status, and α-Myc, to determine Ddc2-AID-myc protein abundance and TIR1-myc as a loading control. (B) Same as (A) for 2-DSB Rad9-AID. (C) Same as (A) for 2-DSB Rad24-AID. (D) Same as (A) for 2-DSB Rad53-AID.

Chk1 is dispensable for activation of the cell cycle arrest, but essential for its maintenance.

(A) Percent G2/M cells in a 2-DSB chk1Δ strain following DNA damage. Data are shown from 3 independent experiments with error bars representing the standard error of the mean (SEM). Western blot probed with α-Rad53 to determine the status of DDC and α-Myc to determine TIR1-myc protein abundance. (B) Adaptation assay with 2-DSB chk1Δ strain. (C) Percentage of G2/M arrested cells a 2-DSB chk1Δ Rad53-AID strain after DNA damage. Cultures were split 4 h after DSB induction and treated with 1 mM auxin (+IAA) or with ethanol (Ctrl). Data are shown from 3 independent experiments with error bars representing the standard error of the mean (SEM). Western blot probed with α-Myc for Rad53-AID and TIR1 as a loading control. (D) Same as (C) for 2-DSB chk1Δ Ddc2-AID. Western blot probed with α-Rad53 and α-Myc. α-Rad53 shows both an unphosphorylated protein and multiple phosphorylated species. α-Myc shows Ddc2-AID degradation and TIR1 as a loading control. The asterisk shows when the percentage of large-budded cells returned to pre-damage levels. (E) Same as (D) for 2-DSB chk1Δ Rad9-AID. (F) Same as (D) for 2-DSB chk1Δ Rad24-AID.

Dun1 is not required for checkpoint maintenance

(A) Adaptation assay of 50 G1 cells on a YEP-Gal plate with 2-DSB dun1Δ. G2/M arrest was determined based on cell morphology as shown in Figure 1A. Data is shown from 3 trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc for TIR1 as a loading control. (B) Percent G2/M arrested cells for 2-DSB Dun1-AID after HO induction. Data are shown from 3 trials with standard error of the mean (SEM). Cultures were split 4 h after DSB induction; with auxin (1 mM) (+IAA). Western blot probed with α-Rad53 and α-Myc. α-Rad53 shows both an unphosphorylated protein and multiple phosphorylated species. α-Myc shows Dun1-AID degradation and TIR1 as a loading control. (C) Same as (B) for 2-DSB chk1Δ Dun1-AID. The asterisk marks when the percentage of large-budded cells returned to pre-damage levels.

Ddc2 and Rad53 are dispensable for >24 h checkpoint arrest

(A) Percent G2/M arrested cells for 2-DSB Ddc2-AID after HO induction. Data is shown from 3 trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc. α-Rad53 shows both an unphosphorylated protein and multiple phosphorylated species. α-Myc shows Ddc2-AID degradation and TIR1 as a loading control. (B) Profile of DAPI stained cells in a 2-DSB Ddc2-AID strain after HO induction. Cells were categorized based on cell morphology and number of DAPI signals. (C) Percent G2/M arrested cells for 2-DSB Rad53-AID TIR1(F74G) after HO induction. 5-Ph-IAA was added 4 h after HO induction. Data is shown from 3 trials with standard error of the mean (SEM). Western blot probed with α-Rad53, α-Myc, and α-Pgk1. α-Rad53 shows both an unphosphorylated protein and multiple phosphorylated species. α-Myc shows Rad53-AID degradation. Pgk1 probed as a loading control. (D) Same as (C) where 5-Ph-IAA was added 15 h after HO induction.

Degradation of Mad2 or Mad1 at 15 h releases cells from checkpoint arrest

(A) Percent G2/M arrested cells for 2-DSB Mad2-AID after HO induction. Data is shown from 3 trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc. α-Rad53 shows both an unphosphorylated protein and multiple phosphorylated species. α-Myc shows Mad2-AID degradation and TIR1 as a loading control. (B) Profile of DAPI stained cells in a 2-DSB Mad2-AID strain after HO induction. Liquid cultures were split 15 h after HO induction and treated with either IAA or ethanol. Cells were scored based on cell morphology and number of DAPI signals. (C) Same as (A) for 2-DSB Mad1-AID. (D) Same as (B) for 2-DSB Mad1-AID.

Degradation of Bub2 but not Bfa1 at 15 h releases cells from checkpoint arrest

(A) Percent G2/M arrested cells for 2-DSB Bub2-AID after HO induction. Data is shown from 3 trials with standard error of the mean (SEM). Western blot probed with α-Rad53 and α-Myc. α-Rad53 shows both an unphosphorylated protein and multiple phosphorylated species. α-Myc shows Bub2-AID degradation and TIR1 as a loading control. (B) Profile of DAPI stained cells in a 2-DSB Bub2-AID strain after HO induction. Liquid cultures were split 15 h after HO induction and treated with either IAA or ethanol. Cells were scored based on cell morphology and number of DAPI signals. (C) Same as (A) for 2-DSB Bfa1-AID. (D) Same as (B) for 2-DSB Bfa1-AID.

Activation and maintenance of checkpoint arrest in response to a DSB

The Mre11-Rad50-Xrs2 (MRX) complex is one of the first complexes recruited to DSBs and initiates the resection of dsDNA to ssDNA. ssDNA is then coated with RPA which recruits Ddc2. Mec1 is the primary kinase responsible for checkpoint arrest in budding yeast and is activated by Ddc2 and Ddc1 from the 9-1-1 clamp. Proteins in green (Ddc2, Rad9, Rad23, and Rad53) were required for the activation and maintenance of checkpoint arrest. While Chk1 was not required for establishment of G2/M arrest, it contributed to the maintenance of arrest. In contrast, Dun1 was required for checkpoint activation but was dispensable 4 h after DSB induction. Prolonged arrest >24 h in a 2-DSB strain was dependent on the SAC proteins Mad2, Mad1, and Bub2 as well as the distance between the 2nd HO-cut site and the centromere.