Diseases, including those related to aging and cancer, often begin with genetic mutations, which can be caused by damage to the genome. Our genome is constantly exposed to sources of damage which may be endogenous, e.g., replication errors, or exogenous, e.g., radiation exposure. Proper identification and repair of DNA damage is therefore essential for cellular health and thus for preventing disease. To help prevent the propagation of mutations, cells have evolved surveillance systems throughout the cell cycle. These surveillance systems, known as checkpoints, arrest the cell cycle when certain conditions are met, such as in the presence of genomic damage. However, after prolonged arrest, many of these checkpoints are overridden, and the cell begins the next cell cycle despite the presence of genomic damage, thus propagating damage to the daughter cell. The choice between staying arrested at checkpoints and allowing time for repair or overriding checkpoints and dividing determines the cell's fate: cells that override checkpoints have a different chance of survival as cells unable to do so. Yet, the biological reasons behind this distinction remain unclear.

To better understand this survival difference and its biological significance, we investigated the consequences of checkpoint override on cellular survival. We used yeast as a model organism, allowing simple genetic manipulations to precisely induce DNA damage and control the cell cycle. By additionally using optogenetics to induce override at different time points after checkpoint arrest, the biological importance of override timing was observed: cell survival rates peaked around the same time as the wild-type override time and slowly decreased when override took place earlier or later.

Subsequently, we studied the mechanism behind the difference in survival of overriding and non-overriding cells. This mechanism could be a purely statistical argument, as at the checkpoint there is only one cell and after override there are two, the mother cell and the new daughter cell, thus increasing the chances of survival. We tested this mechanism by adding a fluorescent tag and following the location of the broken DNA fragments after override. In the majority of cases, we observed that the DNA fragments did not segregate symmetrically between the two cells post-override, and thus, there was only one cell that had all the DNA fragments needed for repair.

Alternatively, the mechanism behind the difference in survival could be due to a new accessibility of various repair pathways after override, thereby increasing the chance of repair. We tested this by using the auxin-inducible degron system to knock down key repair proteins during and after override to determine if that repair protein was required post-override for survival. A dependency on Rad1 and a partial dependence on Dnl4 and Nej1 in overriding cells was observed, suggesting that during or after override, cells rely on microhomology-mediated end joining, an error-prone repair pathway, for repair. \newline \newline Overall, these findings shed light on the biological significance of checkpoint override and uncover a new role for microhomology-mediated end joining, both of which we expect to be highly relevant to expand our knowledge of disease, as malfunctioning checkpoints and erroneous repair can lead to genomic instability.