The Deinococcus protease PprI senses DNA damage by directly interacting with single-stranded DNA

Abstract

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.

Fig. 1. ssDNA physically interactes with PprI.

a Close view of the sulfate and metal ion binding, with HTH motif labeled, sulfate binding residues (Arg85, Arg207, and Arg267) and HEXXH residues (His92, Glu93, and His96) shown as sticks. The electron density of sulfate is shown in blue with the refined 2Fo-Fc contoured at 1σ. b EMSA showing the ssDNA binding of DG-PprI. Proteins (1 or 2 μM) were incubated with 0.1 μM 5’-FAM-labeled ssDNA (35nt), dsDNA (35 bp), or RNA (35nt). c EMSA showing decreased ssDNA binding of the sulfate-binding cavity mutants. 35nt ssDNA (0.1 μM) was incubated with DG-PprI mutant proteins using the same reaction conditions as in panel b. d EMSA assays showing the ssDNA binding of DG-PprI in a length-dependent manner. Proteins were incubated with various length of ssDNA using the same reaction conditions as in panel b. Source data are provided as a Source Data file.

Fig. 2. ssDNA efficiently activates the protease activity of PprI.

a Activation assays showing the ssDNA-enhanced PprI cleavage. DG-DdrO (8 μM) was incubated with DG-PprI (0.1 μM) in the presence of 2 mM MnCl₂ in the absence or presence of 35nt ssDNA (0.1 μM) at 37 °C for 30 minutes. b Quantification of cleavage product of (a). Data are mean ± SD from 3 independent experiments, compared with student unpaired Student’s t test (two-sided). c Metal ion preference of DG-PprI cleavage. DG-PprI (0.1 μM) was incubated with DG-DdrO (8 μM) and ssDNA (0.1 μM) in the presence of 2 mM EDTA or 2 mM divalent metal ions (MnCl₂, MgCl₂, CaCl₂, or ZnCl₂, respectively) at 37 °C for 30 minutes. d ssDNA activation assays containing various lengths (6-10nt) and concentrations (0.01 or 0.1 μM) of ssDNA using the same reaction conditions as in panel a. e Quantifications of cleavage product by various lengths (5, 10, 20, 30, 40nt) and concentrations of ssDNA using the same reaction conditions as in panel a. Data represent the means of the three replicates, and the bars represent their standard deviations. Source data are provided as a Source Data file.

Fig. 3. Interactions between PprI and ssDNA.

a Overall structure of PprI-ssDNA complex. DG-PprI and ssDNA are labeled and shown as cartoon and sticks, respectively. A schematic of the DNA substrate used for crystallization is shown on top with colors corresponding to those observed in the PprI-ssDNA structure below. The electron density of two segments of ssDNA (5ʹ-GCAGTT and 3ʹ-TTTTT) is shown in blue with the refined 2Fo-Fc contoured at 1σ. The catalytic metal ion is shown as sphere and colored red. b The ssDNA binding patches (patches 1-3) of DG-PprI are labeled and shown with electrostatic surface potentials. Blue and red represent the positive and negative charge potential at the + and -5kT e^-1 scale, respectively. c Close view of three ssDNA binding patches of DG-PprI. The EMSA at the bottom-right corner showing the abolished or decreased ssDNA binding of DG-PprI triple mutants (patch 1-3 mutants). The reaction conditions is the same as in Fig. 1c. d Cleavage and ssDNA activation assays of the DG-PprI triple mutants (patch 1-3 mutants). The cleavage assays in the absensece of ssDNA were performed using 1 μM of DG-PprI (lanes 2-5). For ssDNA activation assays (lanes 6-13), 0.1 μM of DG-PprI was used under the same reaction conditions as in Fig. 2a. e Phenotypic analyses of the DG-PprI triple-mutant comlementary strains (patch 1-3 mutants). Wild-type strain (R1), dr_pprI knockout strain (YR1), and dg_pprI complementary strains (YR1-dg_pprI for the wild-type DG-PprI and YR1-patch 1-3 for DG-PprI triple mutants) were spotted on TGY medium following 4 kGy gamma radiation treatments. f Quantitative real-time PCR analysis of the gene expression levels of recA, uvrD, and ddrO. Total RNA was isolated from the R1, YR1, YR1-dg_pprI and YR1-patch1 mutant strains under normal growth conditions and after 8 kGy gamma radiation treatments at different time points during the recovery (15 min, 30 min, 45 min, 1 h and 3 h). Data represent the means of the three replicates, and the bars represent their standard deviations. One-way ANOVA method followed by Tukey’s post-hoc test was performed to compare the significant differences: ***p < 0.001 and ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 4. ssDNA enhances the PprI-DdrO interactions.

a FRET showing the interactions between PprI and DdrO. The transfer efficiency is represented by the RFU ratio of 530/480 nm. Data represent the means of the three replicates and the bars represent their standard deviations. One-way ANOVA method followed by Tukey’s post-hoc test was performed to compare the significant differences. b A Snapshot of all-atom molecular dynamics (MD) simulations of the PprI-DdrO-ssDNA model. The DG-PprI, DdrO, and ssDNA are labeled and shown as surface, cartoon and sticks, respectively. The cleavage center of DG-PprI and the CSR loop of DdrO (Arg118) are highlighted in red and green, respectively. c Close view of cleavage center of PprI and CSR of DdrO during MD simulations. R118 of DdrO interacts with the phosphate group of ssDNA through hydrogen bonds. d RMSD of CSR loop in the ternary complex model during 500 ns simulations. e The relative binding free energy change (ΔΔG) of R118A mutation in the presence of ssDNA was estimated through free energy perturbation (FEP) calculations. Data are presented as mean values +/− SEM from 3 independent experiments. f Cleavage (lanes 1–10) and ssDNA activation (lanes 11-20) assays of the DdrO mutants (E116A, L117A, R118A, and G119A). The reaction conditions is the same as in Fig. 3d. Source data are provided as a Source Data file.

Fig. 5. Dynamic monomer-dimer equilibrium of PprI.

a The left panel shows DG-PprI dimer in a face-to-face fashion in PprI-ssDNA structure. Two DG-PprI protomers are labeled and colored in distinct colors (protomer A in slate and cyan; protomer B in wheat and yellow). The His72 residue of β-pin is labeled and shown as stick. The right panel shows the close view of His72 interactions at the face-to-face dimer interface. b Superposition of two PprI dimer configurations with distinct colors (face-to-face dimer in slate and wheat; side-by-side dimer in black). c Size exclusion chromatography of wild-type (WT) DG-PprI and dimer-interface mutants (2mut: double-mutant at side-by-side interface, 4mut: quadruple-mutant at face-to-face interface, and 6mut: combined double- and quadruple-mutant) on Superdex 200 10/300 GL column. The peaks correspond to monomeric or dimeric PprI proteins are labeled. d Cleavage (lanes 2−5) and ssDNA activation (lanes 6-13) assays of the dimer interface mutants (2mut, 4mut, and 6mut). The reaction conditions is the same as in Fig. 3d. e Phenotypic analyses of the DG-PprI comlementary strains (dimer interface mutants) following 4 kGy gamma radiation treatments. f Quantitative real-time PCR analysis of the gene expression levels of recA, uvrD, and ddrO were performed as in Fig. 3f. Data represent the means of the three replicates, and the bars represent their standard deviations. One-way ANOVA method followed by Tukey’s post-hoc test was performed to compare the significant differences: ***p < 0.001 and ****p < 0.0001. Source data are provided as a Source Data file.