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 init..., 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..., , # 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 tim...