In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

Abstract

The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.

Fig 1. Absence of DdrC increases UV-sensitivity of cells devoid of the UvsE endonuclease.

A Wild type (R1), ΔddrC (GY 15929), ΔuvrA (GY 15971), ΔuvsE (GY 15972), ΔddrC ΔuvrA (GY 15973), ΔddrC ΔuvsE (GY 15974) mutant bacteria grown to an A_650nm = 0.3 were serially diluted in TGY2X broth and aliquots (10 μl) of each dilution were spotted on TGY agar plates. Then, the plates were exposed to UV radiation at the indicated UV doses before incubation at 30°C for 3–5 days. B ΔuvrA ΔuvsE (GY 15977) and ΔddrC ΔuvrA ΔuvsE (GY 15978) and C ΔrecA (GY 15180) and ΔrecA ΔddrC (GY 15965) mutants were treated as described in Fig 1A. All experiments were performed at least 3 times.

Fig 2. The expression of the DdrC protein was induced after exposure to γ-radiation in an IrrE and DdrO dependent manner.

A and B GY15921: ddrC::HA (wt) and GY15967: ddrC::HA ΔirrE (ΔirrE) bacteria exposed or not to 5 kGy γ-radiation were diluted to an A_650nm = 0.2 and incubated at 30°C for the indicated periods (hours). Cell extracts were subjected to SDS-PAGE and analyzed by Western blot with anti-HA antibodies. 5 μg of proteins were loaded on each well. Lane NI: non-irradiated cells. Lane 0 h: non-incubated irradiated cells. C GY16917: a [ddrC::HA ΔddrO (prepU_Ts ddrO⁺)] culture grown at 30°C in TGY2X broth supplemented with spectinomycin (A_650nm = 0.3) was divided into two identical vials and incubated at 30°C or at 37°C, respectively for the indicated periods (hours). Cell extracts were subjected to SDS-PAGE and analyzed by Western blotting with anti-HA antibodies. Ten μg of proteins were loaded on each well.

Fig 3. Cellular localization of DdrC-GFP after γ-irradiation of D. radiodurans cells.

Bacteria expressing a DdrC-GFP fusion protein (GY15931) recovering from γ-irradiation (5 kGy) were visualized by fluorescence microscopy at the indicated times of post-irradiation incubation. DNA was stained with DAPI. Overlays of GFP (green) and DAPI (blue) images as well as overlays of Nomarski DIC (grey), GFP and DAPI are shown.

Fig 4. Dimerization of DdrC in solution.

Lane 1: Purified recombinant DdrC-His₆ protein. Lanes 2–6: Increasing concentrations of recombinant DdrC protein (μM) crosslinked with glutaraldehyde. Lane 7: Molecular weight markers (kDa).

Fig 5. DdrC binds to ssDNA and dsDNA with a preference for ssDNA.

A Binding of recombinant DdrC to plasmid or viral DNA analyzed by EMSA. 200 ng of supercoiled or linear pBR322 DNA as well as 200 ng of RFI or single-stranded DNA of phiX174 virion (31 μM nucleotides of each DNA) were incubated with increasing concentrations of DdrC as indicated in the figure. DNA-protein complexes were separated in 1.2% agarose gels. Products loaded in the right lane of the left panel were treated with SDS and proteinase K. sc: supercoiled dsDNA, oc: open circle dsDNA, Li: linear dsDNA. B Binding of DdrC to oligonucleotides. Increasing concentrations of DdrC were incubated with 3.3 nM of a single-stranded (ss) 67-mer fluorescent oligonucleotide (left panel) or 3.3 nM of the corresponding ds oligonucleotide (right panel). The products of the reactions were separated in 6% native polyacrylamide gels. Lanes C: DNA control without DdrC.

Fig 6. DdrC protects DNA against degradation by nucleases.

Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

Fig 7. DdrC stimulates DNA annealing.

Kinetics of two complementary 67-mer oligonucleotides annealing in the absence (w/o protein) or the presence of DdrC, T4 gp32 or SSB using a DAPI fluorescence-based method. The 67-mer oligonucleotide (200 nM) was mixed in 1 ml of reaction buffer with 0.2 μM DdrC protein, or 0.1 μM T4 gp32, or 0.1 μM SSB from E. coli prior to addition of the reverse oligonucleotide. The extent of DNA annealing is defined as follows: (observed fluorescence—67-mer ssDNA fluorescence) x 100 / 67-mer ds DNA fluorescence.

Fig 8. Visualization of DdrC-DNA complexes by transmission electron microscopy.

A PhiX174 ssDNA (1.4 nM, 7.5 μM nucleotides) was incubated with 1 μM (panels b-d) or 2 μM (panels f-h) of DdrC. Panel a: phiX174 ssDNA control without DdrC. Panel e: Interaction of E. coli SSB protein (1 μM) with ssDNA. Magnification = 85,000. B Supercoiled pBR322 DNA (1.7 nM, 7.5 μM base pairs) incubated with 1 μM (panel b and c) or 2 μM (panel d) of DdrC. Panel a: pBR322 DNA control without protein. Magnification = 85,000. Some“bridge” structures, forming loops or kinks, are indicated by arrows.

Fig 9. Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy.

Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.