TORC2 is required to maintain genome stability during S phase in fission yeast

Abstract

DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.

Keywords: DNA Damage Response; Fission Yeast; Molecular Cell Biology; Rapamycin; S. pombe; Signal Transduction; Stress; TORC2; Yeast Genetics.

FIGURE 1.

The TORC2-Gad8 pathway is required for survival under chronic replicative stress. A–C, 10-fold serial dilution of logarithmic growing cells was plated onto YE-rich medium, containing the indicated DNA-damaging agents or irradiated by UV or IR. Plates were incubated at 30 °C for 3–4 days.

FIGURE 2.

TORC2-Gad8 is required for deactivation of the DNA damage checkpoint but not for its activation. A and B, Tor1 or Gad8 are dispensable for maintaining cellular viability in response to short exposure to MMS (A) or CPT (B). Cells were grown to log phase and shifted to 0.03% MMS or to 30 μm CPT for 6 h. Samples were taken every hour to determine cell viability by assessing plating efficiency on rich medium. C, logarithmic growing cells were transferred to rich medium containing either 30 μm CPT or 0.03% MMS. Samples of the indicated strains, before and after a 6-h exposure to the drugs, were stained with Hoechst and Calcofluor to visualize nuclei and septa, respectively. D, Tor1 is dispensable for Chk1 phosphorylation in response to CPT. Wild type or Δtor1 cells containing an HA-tagged Chk1 were grown to log phase and treated with 30 μm CPT. Protein extract from each of the indicated time points was analyzed by Western blot. The resulting membranes were probed with anti-HA to visualize Chk1 and with anti-Cdc2 as a loading control. E, the dephosphorylation of Chk1 is delayed in a Δtor1 mutant. Cells were arrested for 3 h in 30 μm CPT, washed thoroughly, and resuspended in fresh medium. Protein samples from the indicated time points were analyzed by Western blot as described above.

FIGURE 3.

Elevated levels of Rad52-YFP foci in TORC2-Gad8 mutants. A and B, spontaneous Rad52-YFP foci occur in Δtor1, Δste20, or Δgad8 cells. Cells were cultured in YE liquid medium at 30 °C until mid-log phase, stained with Hoechst and Calcofluor, and visualized live by confocal microscopy. The numbers represent the mean values of three independent experiments, with error bars representing the S.D. Bar, 20 μm. C, quantification of Rad52-YFP foci in nuclei representing different phases of the cell cycle as estimated from cellular and nuclear morphology. D, the assembly and disassembly of Rad52-YFP foci during a transient exposure to CPT. Cells were grown to mid-log phase and arrested in CPT. After 3 h, cells were collected and resuspended in fresh YE medium. Cells were photographed live, at the indicated time points, and the Rad52-YFP foci were counted as described above.

FIGURE 4.

Spontaneous recombination rates are reduced in TORC2-Gad8 mutants compared with wild type. A, schematic representation of the recombination substrate and of the two types of the resulting Ade⁺ recombinants. Gene conversion results in Ade⁺ His⁺, whereas deletion leads to the formation of Ade⁺ His⁻ colonies. B, recombination frequencies of the indicated strains (per 10⁴ cells). Conversion types (black) or deletion types (gray) were determined by replica plating. C, genetic interactions between mutations in TORC2-Gad8 components and deletion of Brc1. 10-fold serial dilutions of logarithmic growing cells were platted on YE-rich medium, containing the indicated concentrations of MMS or CPT. Plates were incubated at 30 °C for 3–4 days.

FIGURE 5.

TORC2-Gad8 mutants show synthetic sick interactions with components of the homologous recombination pathway. A and B, tetrad analysis of heterozygous diploids with the indicated mutations. Representative spores from five asci are shown for each cross. C, strains were grown to log phase in rich medium and visualized by light microscopy. Bar, 20 μm. D and E, 10-fold serial dilution of logarithmic growing cells were plated on YE-rich medium in the presence or absence of the indicated DNA-damaging agents and incubated at 30 °C for 3–4 days.

FIGURE 6.

Genetic interactions between TORC2 and the Swi1-Swi3 fork replication protection complex. A, mutations in tor1⁺ and swi1⁺ show epistasis with respect to spontaneous Rad52-YFP foci level. Strains growing logarithmically in rich medium were stained with Hoechst and imaged live. The percentage of cells containing Rad52-YFP foci was calculated as described in the legend to Fig. 3. B, Tor1 and Swi1 show additive sensitivity to replication stress.