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, 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.