Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint

Reviewer #1 (Public Review):

Summary:
In their manuscript, Zhou et al. analyze the factors controlling the activation and maintenance of a sustained cell cycle block in response to persistent DNA DSBs. By conditionally depleting components of the DDC using auxin-inducible degrons, the authors verified that some DDC proteins are only required for the activation (e.g., Dun1) or the maintenance (e.g., Chk1) of the DSB-dependent cell cycle arrest, while others such as Ddc2, Rad24, Rad9 or Rad53 are required for both processes. Notably, they further demonstrate that after a prolonged arrest (>24 h) in a strain carrying two DSBs, the DDC becomes dispensable and the mitotic block is then maintained by SAC proteins such as Mad1, Mad2, or the mitotic exit network (MEN) component Bub2.

Strengths:
The manuscript dissects the specific role that different components of the DDC and the SAC have during the induction of a cell cycle arrest induced by DNA damage, as well as their contribution to the short-term and long-term maintenance of a DNA DSB-induced mitotic block. Overall, the experiments are well described and properly executed, and the data in the manuscript are clearly presented. The conclusions drawn are also generally well supported by the experimental data. The observations contribute to drawing a clearer picture of the relative contribution of these factors to the maintenance of genome stability in cells exposed to permanent DNA damage.

Weaknesses:
The main weakness of the study is that it is fundamentally based only on the use of the auxin-inducible degron (AID) strategy to deplete proteins. This is a widely used method that allows a very efficient depletion of proteins. However, the drawback is that a tag is added to the protein, which can affect the functionality of the targeted protein or modify its capacity to interact with others. In fact, three of the proteins that are depleted using the AID systems are shown to be clearly hypomorphic. Verification of at least some of the results using an alternative manner to eliminate the proteins would help to strengthen the conclusions of the manuscript.

Reviewer #2 (Public Review):

Summary:
The manuscript analyzes the genetic requirement for DNA damage-induced cell cycle checkpoint induction and maintenance in budding yeast bearing one or two unrepairable DNA double-strand breaks using auxin-induced degradation (AID) of key DNA damage response (DDR) factors. The study paid particular attention to solving a puzzle regarding how yeast bearing two unrepaired DNA breaks fail to engage in "adaptation" whereas those with a single unrepairable break eventually resume cell cycling after a prolonged (up to 12 h) G2 arrest.

The most novel findings are: 1. The genetic requirement for the entry to DDC and the maintenance are separable. For instance, Dun1 is partially required for the entry but not DDC maintenance whereas Chk1 is only required for maintenance. 2. Cells with two irreparable breaks respond to DDR only up to a certain time (~12 h post damage) and beyond this point, depend on spindle assembly checkpoint (SAC) and mitotic exit network (MEN) to halt cell cycling. 3. The authors also propose an interesting model that the location of DNA breaks and their distance to centromeres can lead to the triggering of SAC/MEN and dictate the duration of cell cycle arrest and their adaptability following DNA damage. The results thus provide the most compelling evidence on the role of SAC/MEN in DNA damage response and cell cycle arrest albeit its impact might be limited to the current experimental set-up or under conditions when DNA repair is severely deficient.

Overall, the conclusion of the study is well supported by the elegant set of genetic experimental data and employed multiple readouts on DDC factor depletion on checkpoint integrity and cell cycle status. However, the study still relies heavily on Rad53 phosphorylation as the primary metric to assess checkpoint status. Since evidence exists the residual DDC still operates even when Rad53 phosphorylation is undetectable, additional readouts for DDC functions might be necessary to strengthen the study's conclusions. These and other concerns that need clarifications or further experimental validations are discussed below.

Reviewer #3 (Public Review):

Summary:
The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:
The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1, and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs.

Weaknesses:
Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. In addition, some conclusions about the role of specific DDC proteins in cell cycle arrest at G2/M need further experimental support. The results with Bfa1/Bub2 are surprising and somewhat unexpected. There is no clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block.