Abstract
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 and Rad53; however, after 15 hours both 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
In this updated manuscript we simplified our morphology assay for measuring checkpoint arrest, showed that Rad53-AID is dispensable for prolonged arrest after the handoff of checkpoint arrest from the DNA damage checkpoint (DDC) to the spindle assembly checkpoint (SAC), and that the location of the second HO-cut site relative to the centromere affects whether a strain will remain arrested 24h after DSB induction. Previously we measured the percentage of large-budded G2/M arrested cells in a YEP-Lac+Gal liquid culture and on a YEP-Gal plate. We decided to switch to only using the YEP-Gal plates for our morphology assays because this proves to be a more accurate way to measure escape from checkpoint arrest based on cell morphology. To show that Rad53-AID was not required for prolonged arrest after the handoff, we utilized the new AID2 system to degrade Rad53-AID. Previously we found that due to basal degradation of Rad9-AID, Rad24-AID, and Rad53-AID in the presence of the E3 ligase TIR1 these mutants adapted by 24h. The AID2 system includes a point mutant of TIR1 (TIR1-F74G) and uses 5-phenyl-IAA (5-Ph-IAA) to reduce the basal degradation of AID-tagged proteins. We found that Rad9-AID and Rad24-AID still adapted in the AID2 system, but Rad53-AID remained arrested 24h after DSB induction. Using the AID2 system, we showed that degradation of Rad53-AID 15h after DSB induction did not trigger a release from checkpoint arrest, suggesting that Rad53, much like Ddc2, is not required for prolonged checkpoint arrest after the handoff of checkpoint arrest from the DDC to the SAC. We tested how the distance between the HO-cut site and the centromere affected the prolonged arrest seen in 2-DSB strains. In contrast to the 2-DSB strain we used for this study; it was recently shown that some 2-DSB strains will escape arrest through adaptation 24h after DSB induction. Using modified versions of the well-characterized strain JKM179 which has an HO endonuoclease cleavage site in the MAT locus on chromosome III, we tested the effect of the distance between a second HO cut site on a different chromosome and its centromere on the duration of checkpoint arrest. We found that when the second HO-cut site was moved much further away from the centromere (230kb vs 42/32kb) that cells would experience a prolonged checkpoint arrest compared to the 1-DSB strain JKM179, but over 50% of cells would have adapted 24h after DSB induction.
