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.