DNA damage checkpoint triggers autophagy to regulate the initiation of anaphase

Abstract

Budding yeast cells suffering a single unrepaired double-strand break (DSB) trigger the Mec1 (ATR)-dependent DNA damage response that causes them to arrest before anaphase for 12-15 h. Here we find that hyperactivation of the cytoplasm-to-vacuole (CVT) autophagy pathway causes the permanent G2/M arrest of cells with a single DSB that is reflected in the nuclear exclusion of both Esp1 and Pds1. Transient relocalization of Pds1 is also seen in wild-type cells lacking vacuolar protease activity after induction of a DSB. Arrest persists even as the DNA damage-dependent phosphorylation of Rad53 diminishes. Permanent arrest can be overcome by blocking autophagy, by deleting the vacuolar protease Prb1, or by driving Esp1 into the nucleus with a SV40 nuclear localization signal. Autophagy in response to DNA damage can be induced in three different ways: by deleting the Golgi-associated retrograde protein complex (GARP), by adding rapamycin, or by overexpression of a dominant ATG13-8SA mutation.

Fig. 1.

Vps51 is required for adaptation to the DNA damage checkpoint. WT (JKM179) and vesicular transport mutants were tested in the checkpoint adaptation assay (Materials and Methods). Of these, WT cells adapt whereas vps51∆, vps53∆, and ypt6∆ were found to be adaptation defective. vps52Δ has a similar effect but was measured only at 24 h and is not shown for clarity. Retromer mutant (vps35∆) and ESCRT II mutant (vps25∆) were not adaptation defective. The rdh54∆ cells serve as positive control for the adaptation defect. Cells were pregrown in YEP-Lactate (YEPL) media overnight and then manipulated onto minimal complete media supplemented with 1% yeast extract and containing 2% galactose (CYG). The percentage of cells progressing beyond the “dumbbell” arrest stage (“percentage of adaptation”) is shown.

Fig. 2.

(A and B) pep4∆ and prb1∆ can rescue the adaptation defect in vps51∆ cells but not in rdh54∆, srs2Δ, and ptc2∆ ptc3Δ cells.

Fig. 3.

Pds1 and Esp1 are mislocalized in the cells arrested by DNA damage. (A) Localization of Esp1 and Pds1 during DSB-induced G2/M arrest. The cells were arrested in G2/M phase for 6 h after galactose induction of HO endonuclease. Images were taken without fixation. (B) Quantitation of the images shown in A. The percentage of cells displaying nuclear GFP signal was calculated in each case by counting 100 G2/M-arrested cells per sample. (C) Western blotting of Pds1-GFP and Esp1-myc in the cells arrested by DNA damage. Yeast lysates were prepared 12 h after galactose induction of HO endonuclease. Western blotting of Rho1 is shown as a loading control. Esp1 protein level was not affected 6 h after the DNA DSB. The anti-PSTAIR antibody was used as a reference control.

Fig. 4.

Prevention of vacuolar degradation by pep4Δ reveals that Pds1-GFP can be targeted to vacuole/endosomes. The cells were arrested in G2/M phase for 6 h after galactose induction of HO endonuclease. Pds1-GFP signal intensity was pseudocolored to reflect their signal intensity. These samples were prepared, taken at the same time in the same condition, and the images were equally processed. Arrows point to the position of vacuoles.

Fig. 5.

Artificial nuclear targeting of Esp1 rescues the adaptation defect of vps51Δ. Adding the nuclear localization signal (NLS) from SV40 to the C terminus of Esp1 suppresses the adaptation defect of vps51Δ and partially of srs2Δ but has no effect on two other adaptation-defective mutations.

Fig. 6.

Inhibition of autophagy rescues the adaptation defect of vps51Δ. (A) Deletion of ATG5 suppresses vps51Δ but has little effect on other adaptation-defective mutants. (B) Deletion of ATG1 and ATG11 but not ATG17 suppresses the adaptation defect of vps51Δ. ** denotes statistically significant difference compared with vps51Δ cells. Error bars reflect SEM of three independent experiments.

Fig. 7.

(A) Induction of autophagy prevents adaptation in WT cells. Adaptation assay was performed as described in Materials and Methods except that strains harboring plasmids were grown in selective media containing raffinose overnight to maintain the plasmid and then unbudded cells were spread onto YEP-Galactose plates and monitored for adaptation at 24 h. ** denotes a statistically significant difference compared with the WT strain overexpressing ATG13-8SA. Error bars reflect SEM of three independent experiments. (B) The Rad53 checkpoint kinase is dephosphorylated in GARP mutants with WT kinetics. The indicated strains were grown in YEP-Lactate and DNA damage was induced by adding galactose to the cultures. Samples were taken at various time points denoted and Western blot was performed as described in Materials and Methods.

Fig. 8.

Overexpression of ATG13-8SA causes mislocalization of Pds1-GFP and Esp1-GFP. The images were taken as described in Fig. 3A. Representative images are shown in Fig. S6. The graph denotes the percentage of G2/M-arrested cells that showed nuclear localization of GFP 6 h after galactose induction. At least 100 G2/M-arrested cells were counted per sample.

Fig. 9.

Overexpression of ATG13-8SA (here designated ATG13*) and vps51Δ prevents recovery from DNA damage. WT (YMV80), vps51∆, cdc5-ad, and YMV80+pGAL ATG13-8SA were pregrown in YEP-Lactate and equal numbers of cells were spread onto YEPD plates or YEP-Gal plates. The ratio of the number of viable colonies on YEP-Gal vs. YEPD is presented as percentage of recovery (solid bars). Cells were pregrown in YEP-Lactate liquid media and HO expression was induced with galactose for 6 h. Cells were then spread onto YEP-Gal plates and the cell cycle progression of individual cells was monitored at 24 h after induction. The percentage of cells that have not yet recovered and are still arrested at the two-cell body “dumbbell” stage is presented as percentage of dumbbells (shaded bars). (B) The block in adaptation imposed by overexpression of ATG13* is suppressed by deleting ATG1 or ATG11.

Fig. P1.

Increased autophagy causes the delocalization of both Pds1 (securin) and Esp1 (separase) from the nucleus in checkpoint-arrested budding yeast cells. (A) GFP-tagged Pds1 and Esp1 localize to the nucleus at the bud neck of G2/M-arrested wild-type (WT) cells that have suffered a single unrepaired chromosome double-strand break (DSB). Both rdh54Δ and vps51Δ prevent cells from adapting and resuming cell cycle progression, but ablating only Vps51—part of the Golgi-associated retrograde protein (GARP) complex—causes the mislocalization of Pds1 and Esp1 and the partial degradation of Pds1 by vacuolar proteases. Preventing degradation of Pds1 (and possibly other mitotic regulators) results in the suppression of permanent arrest and the relocalization of sufficient Esp1 into the nucleus to release cells from their preanaphase arrest. A similar suppression of arrest in vps51Δ cells is obtained by disabling autophagy by deleting ATG1 or other genes (not shown). (B) Induction of autophagy by overexpression of ATG13-8SA (5) prevents adaptation in wild-type cells. Expression of ATG13-SA was induced at the same time that a single unrepairable DSB was created. Whereas normal cells adapt by 24 h, increased autophagy prevents most cells from progressing beyond the G2/M stage of the cell cycle. Deletion of the PEP4 gene that activates vacuolar proteases or ATG1 that is required for autophagy suppresses the arrest and allows cells to divide and resume cell cycle progression.