DdrO is an XRE family transcription repressor that, in coordination with the metalloprotease PprI, is critical in the DNA damage response of Deinococcus species. Here, we report the crystal structure of Deinococcus geothermalis DdrO. Biochemical and structural studies revealed the conserved recognizing α-helix and extended dimeric interaction of the DdrO protein, which are essential for promoter DNA binding. Two conserved oppositely charged residues in the HTH motif of XRE family proteins form salt bridge interactions that are essential for promoter DNA binding. Notably, the C-terminal domain is stabilized by hydrophobic interactions of leucine/isoleucine-rich helices, which is critical for DdrO dimerization. Our findings suggest that DdrO is a novel XRE family transcriptional regulator that forms a distinctive dimer. The structure also provides insight into the mechanism of DdrO-PprI-mediated DNA damage response in Deinococcus.

Bacteria have evolved various response systems to adapt to environmental stress. A protease-based derepression mechanism in response to DNA damage was characterized in Deinococcus, which is controlled by the specific cleavage of repressor DdrO by metallopeptidase PprI (also called IrrE). Despite the efforts to document the biochemical, physiological, and downstream regulation of PprI-DdrO, the upstream regulatory signal activating this system remains unclear. Here, we show that single-stranded DNA physically interacts with PprI protease, which enhances the PprI-DdrO interactions as well as the DdrO cleavage in a length-dependent manner both in vivo and in vitro. Structures of PprI, in its apo and complexed forms with single-stranded DNA, reveal two DNA-binding interfaces shaping the cleavage site. Moreover, we show that the dynamic monomer-dimer equilibrium of PprI is also important for its cleavage activity. Our data provide evidence that single-stranded DNA could serve as the signal for DNA damage sensing in the metalloprotease/repressor system in bacteria. These results also shed light on the survival and acquired drug resistance of certain bacteria under antimicrobial stress through a SOS-independent pathway.

RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5′-P-dSpacer group, a 5′-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5′-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5′-monophosphate binding pocket and occupancy of 5′-dRP in the drRecJ nuclease core. The mechanism for RecJ 5′-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5′-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.

DNA-damage chemicals, including many antibiotics, often induce prophage induction and phage outbreaks within microbial communities, posing a significant threat to bacterial survival. Moraxellaceae strains are clinically relevant due to their remarkable resistance to antibiotics and radiation. However, the cellular-level regulation mechanisms that underlie their DNA damage response and anti-phage defense remain extensively unexplored. Here, we report a WYL family protein, DdaA, that has replaced the ubiquitous SOS system during the evolution of Moraxellaceae. DdaA functions as an activator and directly regulates the transcriptional networks of both DNA damage response and anti-phage defense genes under conditions of DNA damage stress. Our findings elucidate a pathway that shows how these bacteria enhance their immunity under DNA damage and shed light on controlling the resistance of Moraxellaceae strains in clinical practice.

DNA damage occurs when cells encounter exogenous and endogenous stresses such as long periods of desiccation, ionizing radiation and genotoxic chemicals. Efforts have been made to detect DNA damage in vivo and in vitro to characterize or quantify the damage level. It is well accepted that single‐stranded DNA (ssDNA) is one of the important byproducts of DNA damage to trigger the downstream regulation. A recent study has revealed that PprI efficiently recognizes ssDNA and cleaves DdrO at a specific site on the cleavage site region (CSR) loop in the presence of ssDNA, which enables the radiation resistance of Deinococcus . Leveraging this property, we developed a quantitative DNA damage detection method in vitro based on fluorescence resonance energy transfer (FRET). DdrO protein was fused with eYFP and eCFP on the N‐terminal and C‐terminal respectively, between which the FRET efficiency serves as an indicator of cleavage efficiency as well as the concentration of ssDNA. The standard curve between the concentration of ssDNA and the FRET efficiency was constructed, and application examples were tested, validating the effectiveness of this method.