AbstractBacteria 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 geneddcAas critical for surviving exposure to a broad spectrum of DNA damage. TheddcAdeletion phenotypes are dependent on the checkpoint enforcement protein YneA. We found that expression of the checkpoint recovery proteases could not compensate forddcAdeletion. Similarly, expression ifddcAcould not compensate for the absence of the checkpoint recovery proteases, indicating that DdcA function is distinct from the checkpoint recovery step. Deletion ofddcAresulted in sensitivity toyneAoverexpression 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 bacteriumBacillus subtilisis 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.
Transcription–Replication Coordination
