DOI: 10.5061/dryad.sj3tx96dv
title: Data from: Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint

description: 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.
description: <strong>Microscopy, DAPI staining, and Cell Morphology
 Determination</strong>  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 G<sub>2</sub>/M arrested
 cells with large buds. For each time-point, &gt;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. <strong>Adaptation and Auxin Plating
 Assays</strong>   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
 G<sub>1</sub> 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, &gt;250 cells were scored and categorized as
 described above for the adaptation assay from at least 3 biological
 replicates. <strong>Quantification and Data
 Analysis</strong> Graphs were prepared using
 GraphPad Prism 10 (Dotmatics). Statistical analysis for differences in the
 percentage of large budded (G<sub>2</sub>/M arrested) cells at
 different timepoints listed in <strong>Table 1</strong> 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.
description: # Prolonged cell cycle arrest in response to DNA damage in yeast requires
 the maintenance of DNA damage signaling and the spindle assembly
 checkpoint
 [https://doi.org/10.5061/dryad.sj3tx96dv](https://doi.org/10.5061/dryad.sj3tx96dv) ## Description of the data and file structure **Date of Collection** 2016-2024 **Contributors** Felix Y. Zhou1, David P. Waterman, Marissa Ashton, Suhaily Caban-Penix, Gonen Memisoglu, Vinay V. Eapen, and James E. Haber **Overview** These datasets contain the data used to determine the state of checkpoint arrest through the DNA damage checkpoint and spindle assembly checkpoint proteins following a double-stranded break.  **Files and variables** **File: 01_Adaptation Assay.xlsx** File format: xlsx file Description: Data from adaptation assays. In each adaptation assay 50 G1 cells were selected after being placed on a YP-Gal plate. Each tab represents a different strain or set of strains. The number of large-budded (G2/M arrested) cells at each timepoint is presented and the trial number is listed in Column A. Unless otherwise stated all strains were made in a 2-DSB background. Please refer to the Key Resources Table for strain names and genotypes. **File: 02_DAPI.xlsx** File format: xlsx file Description: Data showing the population of DAPI stained cells following double-strand break induction with Gal-HO at different timepoints. Each tab lists the strain name and when IAA was added after double-strand break induction e.g. DW418, 15h is strain DW418 and IAA was added to a split culture 15h after DSB induction. Column A shows the trial number and column B shows whether cells were treated with IAA. The name of each graph shows the strain name, and the genotype can be found in the Key Resources Table. Cells were divided into 4 categories based on cell morphology and the number of DAPI signals. G1 cells had no bud and small budded cells had a small bud growing on the cell. To tell the difference between small budded and large budded cells, a line can be drawn from the bud tip of the mother cell to the bud tip of the daughter cell through the bud neck. If the length from the bud tip to the bud tip of the daughter cell is greater than or equal to 40% the length from tip to tip, then the cell is a large budded cell. Large budded cells with 1 DAPI signal had not passed into anaphase. An accumulation of these cells were considered G2/M arrested. Large budded cells with 2 DAPI signals had proceeded through anaphase and were no longer arrested at the G2/M checkpoint. **File: 03_Ddc2-Levels.xlsx** File format: xlsx file Description: Data from western blots quantifying the relative amount of Ddc2 in cells in a 1-DSB (GM539) and a 2-DSB (DW418) background up to 24 h after DSB induction with Gal-HO. Column A shows the time since Gal-HO induction. Columns B through G show the relative amount of Ddc2 from each trial normalized to the 0 h timepoint before Gal-HO was induced. Ddc2 levels were measured every 3 hours after DSB induction for up to 24 hours. Ddc2 levels were normalized to the 0 hour timepoint before galactose was added. **File: 04_Plating Assay** File format: xlsx file Description: Data showing the percentage of G2/M arrested cells following double-strand break induction. Cells were initially grown in a YP-Lac liquid culture. After adding galactose to induce Gal-HO cutting, cultures were plated on either YP-Gal or YP-Gal-IAA plates. Each tab shows the strain used and the time when they were plated on either YP-Gal or YP-Gal-IAA plates after galactose was added e.g. FZ009, 15h is strain FZ009 and they were plated after growing in YP-Lac+Gal for 15 hours. Column A shows the trial number and column B shows whether they were plated on YP-Gal or YP-Gal+IAA plates. Please refer to the Key Resources Table for strain names and genotypes. **File: 05_Histone Growth Data.xlsx** File format: xlsx file Description: Data showing the doubling times of H2A and H2B histone point mutants. H2A was mutated at serine 129 (S129) to either alanine (S129A) to make a non-phosphorylated mutant or glutamic acid (S129E) to make a phosphomimetic mutant. H2B was mutated at threonine 129 (T129) to either alanine (T129A) to make a non-phosphorylated mutant or glutamic acid (T129E) to make a phosphomimetic mutant. Cultures were synchronized so they all started with similar densities as measured by optical density at 600nm (OD600). Cell density was measured at 3, 5, 7, and 10 hours after cultures were synched. Please refer to the Key Resources Table for strain names and genotypes. **File: 06_IAA Before Gal.xlsx** File format: xlsx file Description: Data showing the percentage of large budded (G2/M arrested) cells where IAA was added 1 h before double-strand break induction with Gal-HO in AID-tagged mutants. The tab indicates the strain tested. Column A shows the trial number. IAA was added 1 h before adding galactose in all strains. Please refer to the Key Resources Table for strain names and genotypes. **File: Key Resource Table** File format: xlsx file Description: Key Resource Table that includes descriptions of the strains used, genes studied, plasmids antibodies, reagents, and kits used. Please refer to this table for strain information.  **Code/software** Microsoft Excel was used to view Excel files.