Using optogenetics to find the signal that maintains the DNA damage checkpoint

Cell cycle checkpoints arrest the cell division cycle when certain types of damage such as double-strand DNA breaks (DSBs) are detected. However, after prolonged arrests, many checkpoints are eventually overridden when damage persists. We recently studied the DNA damage checkpoint theoretically and experimentally in budding yeast and found that DDC override strikes a balance between speed and risk (Sadeghi et al., Nature Physics 2022). However, the nature of the signal maintaining the checkpoint has not been known. For the DDC, two different signals have been proposed in the past, emanating from the tips of broken chromosomes or from single-strand DNA (ssDNA) that is resected around the DNA break. To test which model is correct, we measured the rate of DNA resection using single-cell probes. We integrated light-activated promoter-fluorescent protein gene constructs at specific intervals from DNA break sites. By detecting when their activity was silenced, we could observe how much DNA had been resected. Using these measurements for wild-type and resection-defective strains, we found that a mixed model fit the data best: Both resected DNA as well as DNA break ends make distinct contributions to maintaining the DDC arrest. Furthermore, one DNA break affects resection of another, indicating interactions between DSBs.