Prolonged DNA Damage Checkpoint Arrest Requires Constant Renewal and the Spindle Assembly Checkpoint.

To prevent cell division in the presence of a DNA double-strand breaks (DSB), cell cycle progression is arrested by the DNA damage checkpoint (DDC) to allow more time for repair. In budding yeast, a single irreparable DSB arrests cells for about 12 h - 6 normal doubling times - after which cells adapt to the damage and resume the cell cycle. In contrast, 2 DSBs provoke permanent G2/M arrest. While activation of the DDC is well-understood, how it is maintained remains unclear. To address this question, key checkpoint proteins were inactivated by auxin-inducible degradation 4 h after damage induction. Degradation of Ddc2 ATRIP , Rad9, Rad24, or Rad53 CHK2 resulted in resumption of cell cycle, indicating that these checkpoint factors are required both to establish and to maintain DDC arrest. However, when Ddc2 is inactivated 15 h after inducing 2 DSBs, cells remain arrested. This continued arrest depends on the spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2. Although Bub2 acts with Bfa1 to regulate mitotic exit, inactivation of Bfa1 did not trigger checkpoint release. 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.