DdcA antagonizes a bacterial DNA damage checkpoint

Abstract

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.

Regulation of Cell Division in Bacteria by Monitoring Genome Integrity and DNA Replication Status

“…3 and Table 1) (112). The same Tn-seq screen that discovered DdcP and CtpA also found a cytoplasmic protein DdcA that negatively regulates YneA helping to set the threshold of YneA necessary to induce the checkpoint (113). The discovery of DdcA strengthens the evidence that bacterial cells have multiple mechanisms to ensure that enough DNA damage must be detected before engaging the checkpoint.…”

DNA damage checkpoint activation affects peptidoglycan synthesis and late divisome components in Bacillus subtilis

“…As a result, we expect a cell proliferation defect following MMC treatment because endogenous yneA should be activated. When we treat cells with increasing concentrations of MMC, we find that cells are sensitive to MMC and this phenotype is more severe in the absence of the ddcP, ctpA, and ddcA negative regulators of YneA (Figure 3b) as described (Burby et al, 2019). If this phenotype is due to induction of F I G U R E 2 Isolation of mutations in yneA that prevent checkpoint activation.…”

“…We further investigated the effect of yneA expression on cell proliferation by treating cells with increasing concentrations of the DNA damaging agent mitomycin C (MMC) (Iyer & Szybalski, 1963;Noll et al, 2006) to induce native yneA in the presence or absence of the IPTG regulated yneA allele (Burby et al, 2019;Figure 3b). It was previously shown that YneA inhibits cell division in B. subtilis following DNA damage (Burby et al, 2018(Burby et al, , 2019Kawai et al, 2003). As a result, we expect a cell proliferation defect following MMC treatment because endogenous yneA should be activated.…”

The ins and outs of Bacillus proteases: activities, functions and commercial significance

“…Deletion of the genes encoding CtpA and DdcP leads to the accumulation of the DNA damage checkpoint protein YneA, a small membrane bound inhibitor that delays cell division and thereby provides a time frame during which damaged chromosomes are repaired (Burby et al . 2018 , 2019 ). CtpA and CtpB specifically recognize a C-terminal tripeptide, X-Y-Z, in which X is preferably Ala or Leu, Y is preferably Ala or Tyr, and Z is preferably Ala.…”