The DNA damage response is a signaling pathway found throughout biology. In many bacteria the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair damaged chromosomes. How cells promote checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the checkpoint protein YneA. We show that DNA damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors.

Summary Bacteria coordinate DNA replication and cell division, ensuring a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage, a cell cycle checkpoint is activated by expressing a cell division inhibitor. The prevailing model is that activation of the DNA damage response and protease mediated degradation of the inhibitor is sufficient to regulate the checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to DNA damage. Similar to the checkpoint recovery proteases, the DNA damage sensitivity resulting from ddcA deletion depends on the checkpoint enforcement protein YneA. Using several genetic approaches, we show that DdcA function is distinct from the checkpoint recovery process. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels and stability, further supporting the conclusion that DdcA regulates YneA independent of proteolysis. Using a functional GFP-YneA fusion we found that DdcA prevents YneA-dependent cell elongation independent of YneA localization. Together, our results suggest that DdcA acts by helping to set a threshold of YneA required to establish the cell cycle checkpoint, uncovering a new regulatory step controlling activation of the DNA damage checkpoint in Bacillus subtilis.

Bacteria coordinate DNA replication and cell division, ensuring that a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage or impediments to DNA replication, a cell cycle checkpoint is activated to delay cell division by expressing a cell division inhibitor. The prevailing model for bacterial DNA damage checkpoints is that activation of the DNA damage response and protease mediated degradation of the cell division inhibitor is sufficient to regulate the checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to a broad spectrum of DNA damage. The ddcA deletion phenotypes are dependent on the checkpoint enforcement protein YneA. We found that expression of the checkpoint recovery proteases could not compensate for ddcA deletion. Similarly, expression if ddcA could not compensate for the absence of the checkpoint recovery proteases, indicating that DdcA function is distinct from the checkpoint recovery step. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels or stability, further supporting the conclusion that DdcA regulates YneA through a proteolysis independent mechanism. Using a functional GFP-YneA we found that DdcA inhibits YneA activity independent of YneA localization, suggesting that DdcA may regulate YneA access to its target. These results uncover a regulatory step that is important for controlling the DNA damage checkpoint in bacteria, and suggests that the typical mechanism of degrading the checkpoint enforcement protein is insufficient to control the rate of cell division in response to DNA damage.Author SummaryAll cells coordinate DNA replication and cell division. When cells encounter DNA damage, the process of DNA replication is slowed and the cell must also delay cell division. In bacteria, the process has long been thought to occur using two principle modes of regulation. The first, is RecA coated ssDNA transmits the signal of DNA damage through inactivation of the repressor of the DNA damage (SOS) response regulon, which results in expression of a cell division inhibitor establishing the checkpoint. The second principle step is protease mediated degradation of the cell division inhibitor relieving the checkpoint. Recent work by our lab and others has suggested that this process may be more complex than originally thought. Here, we investigated a gene of unknown function that we previously identified as important for survival when the bacterium Bacillus subtilis is exposed to DNA damage. We found that this gene negatively regulates the cell division inhibitor, but is functionally distinct from the checkpoint recovery process. We provide evidence that this gene functions as an antagonist to establishing the DNA damage checkpoint. Our study uncovers a novel layer of regulation in the bacterial DNA damage checkpoint process challenging the longstanding models established in the bacterial DNA damage response field.