Zhou Felix Y, Waterman David P, Ashton Marissa, Caban-Penix Suhaily, Memisoglu Gonen, Eapen Vinay V, Haber James E
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, United States.
Department of Molecular Genetics & Cell Biology, University of Chicago, Chicago, United States.
Elife. 2024 Dec 10;13:RP94334. doi: 10.7554/eLife.94334.
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 12-15 hr, 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 Ddc2, Rad9, Rad24, or Rad53 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and maintain the arrest. Dun1 is required for the establishment, but not the 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 two persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hr 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 require overlapping but different sets of factors.
在存在DNA双链断裂（DSB）的情况下，细胞会激活DNA损伤检查点（DDC）以抑制有丝分裂，从而为DNA修复留出更多时间。在芽殖酵母中，单个无法修复的DSB足以激活DDC，并在后期之前诱导细胞周期停滞约12 - 15小时，之后细胞通过消除DDC并恢复细胞周期来“适应”损伤。虽然DNA损伤依赖性细胞周期停滞的激活机制已得到充分理解，但其维持机制仍不清楚。为了解决这个问题，我们在DDC完全激活后有条件地耗尽关键的DDC蛋白，并监测细胞周期停滞维持过程中的变化。Ddc2、Rad9、Rad24或Rad53的降解会导致细胞周期过早恢复，这表明这些DDC因子对于建立和维持停滞都是必需的。Dun1是建立停滞所必需的，但不是维持停滞所必需的，而Chk1是延长维持停滞所必需的，但不是有丝分裂停滞初始建立所必需的。当细胞受到两个持续性DSB的挑战时，它们会永久停滞。这种永久停滞最初依赖于Ddc2、Rad9和Rad53的持续存在；然而，15小时后这些蛋白质变得不再必要。相反，持续的有丝分裂停滞由纺锤体组装检查点（SAC）蛋白Mad1、Mad2和Bub2维持，但不由Bub2的结合伴侣Bfa1维持。这些数据表明，对2个DSB的延长细胞周期停滞是通过从DDC交接给SAC的特定组件来实现的。此外，DNA损伤诱导的细胞周期停滞的建立和维持需要重叠但不同的一组因子。
