Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells “adapt” to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hours these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2’s binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

Methods
Microscopy, DAPI staining, and Cell Morphology Determination 

Aliquots from YEP-Lac cultures were taken either 4 h or 15 h after adding galactose, diluted 20-fold in sterile water and plated on a YEP-Agar with 2 % galactose with or without 1 mM IAA or 1 µM 5-Ph-IAA. Cells were counted on a light microscope with a 10x objective, examined and binned into three categories: unbudded, small buds, and G2/M arrested cells with large buds. For each time-point, >250 cells were analyzed. For DAPI staining, 450 µl of culture was added to 50 µl of 37% formaldehyde and incubated in the chemical hood at room temperature for 20 min. Samples were spun down at 8000 rpm for 5 min and washed with 1X PBS 3 times. Cells were resuspended in 50 µl of DAPI mounting media (VECTASHIELD® Antifade Mounting Medium with DAPI H-1200-10) and incubated at room temperature for 10 min, away from direct light. The samples were imaged by using a Nikon Ni-E upright microscope equipped with a Yokogawa CSU-W1 spinning-disk head, an Andor iXon 897U EMCCD camera, Nikon Elements AR software, a 60x oil immersion objective, and a 358 nm laser. 15 z-stacks with a thickness of 0.3 µm were collected per image. In the morphology assays at least 3 biological replicates were used for each strain.

Adaptation and Auxin Plating Assays  

We performed adaptation assays as previously described (Eapen et al. 2012; Lee et al. 1998). Cells grown in YEP-Lac overnight were diluted 20-fold in sterile water and plated on a YEP-agar plate containing 2 % galactose. Using micromanipulation, 50 G1 cells were isolated and positioned in a grid followed by incubation at 30° C. To quantify the percentage of adapted cells, the number of cells that re-entered cell cycle, grew to a microcolony (3+ cells) after 24 h was divided by the total number of cells. For an auxin plating assays, damage was induced in a YEP-Lac liquid culture by adding galactose at a final concentration of 2 %, as described above. Cells were then transferred onto YEP-agar plates containing 2 % galactose and 1mM IAA or 1 µM 5-Ph-IAA 4 h or 15 h after adding galactose. For each timepoint, >250 cells were scored and categorized as described above for the adaptation assay from at least 3 biological replicates.

Quantification and Data Analysis

Graphs were prepared using GraphPad Prism 10 (Dotmatics). Statistical analysis for differences in the percentage of large budded (G2/M arrested) cells at different timepoints listed in Table 1 was done using a one-way Anova test in GraphPad Prism 10. Protein quantification of Ddc2-myc blots was done using ImageLab 6.1 (BioRad). To categorize DAPI stained cells based on their morphology and number of DAPI signals, images were captured as described above and viewed using ImageJ with the Fiji addon.