Deinococcus radiodurans HD-Pnk, a Nucleic Acid End-Healing Enzyme, Abets Resistance to Killing by Ionizing Radiation and Mitomycin C

End healing is a process whereby nucleic acid breaks with “dirty” 3′-PO₄ or 2′,3′-cyclic-PO₄ and 5′-OH ends are converted to 3′-OH and 5′-PO₄ termini that are amenable to downstream repair reactions. Deinococcus radiodurans is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights Deinococcus HD-Pnk as a bifunctional 3′- and 5′-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

ABSTRACT

5′- and 3′-end healing are key steps in nucleic acid break repair in which 5′-OH and 3′-PO₄ or 2′,3′-cyclic-PO₄ ends are converted to 5′-PO₄ and 3′-OH termini suitable for sealing by polynucleotide ligases. Here, we characterize Deinococcus radiodurans HD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5′-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3′-phosphatase and 2′,3′-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis of p-nitrophenylphosphate. HD-Pnk is encoded by the LIG–PARG–HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential for Deinococcus growth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitize Deinococcus to killing by mitomycin C.

IMPORTANCE End healing is a process whereby nucleic acid breaks with “dirty” 3′-PO₄ or 2′,3′-cyclic-PO₄ and 5′-OH ends are converted to 3′-OH and 5′-PO₄ termini that are amenable to downstream repair reactions. Deinococcus radiodurans is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights Deinococcus HD-Pnk as a bifunctional 3′- and 5′-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

INTRODUCTION

The repair of nucleic acid damage from ionizing radiation (IR) and other genotoxic stresses is crucial for genome maintenance (1). IR elicits oxidative damage to nucleic acids, including single-strand and double-strand breaks in the phosphodiester backbone. IR breakage leaves “dirty” 3′-phosphate and 3′-phosphoglycolate ends (2) that must be converted to “clean” ends to facilitate the actions of repair polymerases and repair ligases that restore the phosphodiester backbone. Our goal is to elucidate the mechanisms, structures, and evolutionary relationships of enzymes that heal and seal broken nucleic acid ends and to leverage those insights to discover new types of repair enzymes and new repair pathways in diverse biological contexts.

Bacterial model systems are a foundational source of genetic and biochemical insights to radiation sensitivity and nucleic acid repair (1). Whereas Escherichia coli has been a rich model to delineate conserved core pathways and enzymatic agents of repair, recent studies underscore that E. coli does not reflect the full spectrum of repair mechanisms found in eukarya, or even in other model bacteria. (For example, E. coli does not have a Ku-driven nonhomologous end-joining pathway, operative in eukarya and other bacteria, that aids in the repair of IR damage [3].) Deinococcus radiodurans is a choice model bacterium to probe the basis for resistance to massive doses (in kilograys [kGy]) of gamma radiation that generate hundreds of DNA double-strand breaks and thousands of DNA single-strand breaks (4, 5). Whereas faithful restoration of the radiation-shattered Deinococcus genome depends on several repair proteins and pathways that are conserved in E. coli, it also relies on additional enzymes and factors that are either unique to Deinococcus or are shared with bacteria other than E. coli (5). The premise of our studies is that by defining new enzymes implicated in break repair and interrogating their roles in Deinococcus radiation resistance, we will enrich the understanding of how nucleic acid breaks are rectified.

Toward that end, we previously undertook a biochemical and genetic characterization of Deinococcus RNA ligase (DR_B0094, named DraRnl), which is the founding member of the Rnl5 family of ATP-dependent polynucleotide ligases present in a variety of bacteria, bacterial viruses, eukarya, and archaea (6–10). DraRnl seals 3′-OH/5′-PO₄ nicks in duplex nucleic acids in which the 3′-OH nick terminus consists of two or more ribonucleotides (6). We showed via gene knockout that whereas DraRnl is inessential for the growth of D. radiodurans, its absence sensitizes the bacterium to killing by ≥9-kGy gamma radiation (10). The DraRnl protein is depleted during the early stages of recovery from 10 kGy of IR and subsequently replenished during the late phase of post-IR genome reassembly (10). The absence of DraRnl elicits a delay in reconstitution of the 10-kGy IR-shattered D. radiodurans replicons. These results suggest that DraRnl is deployed for ligation at sites of “ribo-patch” gap repair where ribonucleotides are added to a DNA 3′-OH end by repair polymerases.

In the present study, we turn our attention to the enzymology of “end healing” in Deinococcus, the process whereby dirty 3′-PO₄ and 5′-OH nucleic acid ends are converted to 3′-OH and 5′-PO₄ termini that are amenable to downstream repair reactions. Healing of 5′-OH ends is the province of polynucleotide kinases (Pnks), a widely distributed class of cellular and virus-encoded nucleic acid repair enzymes. Pnks are members of the P-loop phosphotransferase superfamily; they catalyze metal-dependent transfer of the γ-phosphate of a nucleoside triphosphate (NTP) donor to a 5′-OH polynucleotide acceptor. In many repair systems, a Pnk enzyme is fused in a modular fashion to a 3′ end-healing enzyme within a single multifunctional polypeptide (11–17).

This appears to be the case in Deinococcus, as exemplified by the 417-amino-acid (aa) HD-Pnk protein (DR_B0098), which was predicted to be a bifunctional 3′- and 5′-end-healing enzyme composed of an N-terminal HD (histidine-aspartate) phosphoesterase module and a C-terminal kinase module. Blasius et al. (18) showed that recombinant DR_B0098 has 5′-OH DNA kinase activity in the presence of manganese, but they were unable to assign DNA 3′-phosphatase activity to the recombinant protein. Our laboratory recently characterized the homologous 368-aa HD-Pnk protein from the bacterium Runella slithyformis and showed that, in addition to its Pnk activity, HD-Pnk is a 3′-end-healing enzyme that dephosphorylates RNA 2′,3′-cyclic phosphate, RNA 3′-phosphate, RNA 2′-phosphate, and DNA 3′-phosphate ends in the presence of a transition metal cofactor, which can be nickel, copper, or cobalt (19).

As shown in Fig. 1B, the Deinococcus and Runella HD-Pnk polypeptides share 150 positions of side-chain identity/similarity. The C-terminal half of HD-Pnk contains two hallmark motifs (Fig. 1B, cyan) found in polynucleotide kinases: (i) the GXXGXGK “P-loop” that engages the phosphates of the NTP donor substrate via hydrogen bonds from the P-loop lysine Nζ and several of the P-loop main-chain amide nitrogens and (ii) a DXXR motif that engages the 5′-_HONp nucleotide of the phosphate acceptor, such that the aspartate (Asp254 in HD-Pnk) acts as a general base catalyst to activate the O-5′ nucleophile for its attack on the NTP γ-phosphorus, and the arginine coordinates the 3′-phosphate (20). HD domain enzymes are a widely distributed family (21) of binuclear metal-dependent phosphodiesterases and monoesterases that act on nucleic acids, nucleotides, and other phosphate-containing substrates in the service of a broad spectrum of biological functions. The N-terminal half of HD-Pnk contains the signature HDXXK motif (shaded gold in Fig. 1B), which coordinates the metal cofactor(s) via the histidine and aspartate side chains and the scissile phosphoester via the lysine side chain (22, 23).

FIG 1.

Genomic contexts and primary structures of Deinococcus and Runella HD-Pnk. (A) The Deinococcus radiodurans DR_B0098 ORF encoding HD-Pnk is preceded by two cooriented upstream ORFs encoding enzymes with likely roles in nucleic acid repair, a poly(ADP-ribose) glycohydrolase (PARG), and an ATP-dependent polynucleotide ligase (LIG). Runella slithyformis HD-Pnk is encoded in a two-gene cluster that includes an upstream LIG gene. (B) The amino acid sequence of D. radiodurans (Dra) HD-Pnk (NCBI accession no. AAF12617) is aligned to that of R. slithyformis HD-Pnk (Rsl) (NCBI accession no. AEI48611). Positions of side-chain identity/similarity are denoted by dots. Gaps in the alignments are denoted by dashes. A signature HDXXK motif of the HD active site is highlighted in gold shading. The signature residues of the Pnkp P-loop motif (GXXGXGK) that coordinates the NTP phosphates and the DXXR motif that coordinates the 5′ _HONp nucleotide of the phosphoacceptor are highlighted in cyan. The His81, Asp82, and Lys277 residues that were mutated to alanine are denoted by arrowheads.

The prospect of a nucleic acid repair function for Deinococcus HD-Pnk is fortified by its location within a cooriented three-gene cluster in the D. radiodurans genome that encodes two other enzymes with likely roles in nucleic acid repair, a poly(ADP-ribose) glycohydrolase (PARG) (24) and an ATP-dependent polynucleotide ligase-like adenylyltransferase (LIG) (18), in a putative operon in which the 3′ end of the PARG open reading frame (ORF) overlaps the 5′ end of the HD-Pnk ORF (Fig. 1A). Moreover, this LIG–PARG–HD-Pnk operon is transcriptionally upregulated after exposure of D. radiodurans to 15 kGy of IR (25, 26). Runella slithyformis HD-Pnk is encoded by a two-gene operon-like cluster with an upstream LIG gene (Fig. 1A) (19). Because HD-Pnk homologs are present in genera from 11 bacterial phyla and are often encoded in an operon with a LIG gene (19), we speculate that the end-healing function of HD-Pnk and the imputed end sealing function of LIG are functionally connected.

In the present study, we purified recombinant Deinococcus HD-Pnk and documented its dual kinase and phosphoesterase activities, which are mediated by the Pnk and HD active sites, respectively. We report that deletion of the HD-Pnk gene sensitizes Deinococcus to killing by IR. We find that (i) HD-Pnk is depleted within 3 h after 9 kGy of IR exposure and is replenished at 15 h post-IR, subsequent to reassembly of the genome replicons from shattered DNA fragments, and (ii) the absence of HD-Pnk does not prevent genome reassembly, as monitored by pulsed-field gel electrophoresis. Active-site mutations of HD-Pnk also sensitize Deinococcus to killing by mitomycin C. We discuss potential roles for HD-Pnk in nucleic acid damage repair.

RESULTS

Polynucleotide kinase activity of recombinant HD-Pnk.

To assess what biochemical activities, if any, are associated with Deinococcus HD-Pnk, we produced and purified recombinant wild-type HD-Pnk and two HD-Pnk mutants in which alanine replaced predicted Pnk active-site residue Lys277 or the HD motif residues His81 and Asp82 (Fig. 2A). Polynucleotide kinase activity of wild-type HD-Pnk was demonstrated by incubating the protein with magnesium, [γ-³²P]ATP, and a 36-mer 5′-OH DNA oligonucleotide (Fig. 3A). This resulted in label transfer from ATP to the 36-mer DNA, as gauged by denaturing PAGE and autoradiography (Fig. 3A). DNA phosphorylation required a divalent cation. A comparison of magnesium, manganese, calcium, cadmium, cobalt, copper, nickel, and zinc at 5 mM concentration showed that magnesium and manganese were the preferred metal cofactors (Fig. 3A). Cobalt and nickel were less effective. Calcium supported barely detectable kinase activity. Cadmium, copper, and zinc were inactive (Fig. 3A). Superdex-200 gel filtration showed that the 45-kDa HD-Pnk polypeptide eluted as a single component at a position relative to native size standards consistent with it being a monomer in solution (Fig. 4A). An assay of Pnk activity of the even-numbered Superdex column fractions (Fig. 4B), in parallel with SDS-PAGE analysis of the same fractions (not shown), affirmed that kinase activity coeluted with the HD-Pnk protein during gel filtration and peaked in column fraction 36 (Fig. 4B). The kinase activity was effaced by the K277A mutation in the Pnk P-loop motif but was not affected by the H81A-D82A mutation of the HD motif (Fig. 2B).

FIG 2.

Recombinant HD-Pnk and mutational effects on kinase and phosphatase activity. (A) Aliquots (4 μg) of wild-type HD-Pnk (WT) and the H81A-D82A and K277A mutants were analyzed by SDS-PAGE. The Coomassie blue-stained gel is shown. The positions and sizes (in kilodaltons) of marker polypeptides are indicated on the left. The HD-Pnk polypeptide is denoted by an arrow. (B) Kinase assay. Reaction mixtures (10 μl) containing 50 mM Tris-acetate (pH 6.5), 2 mM DTT, 5 mM MgCl₂, 50 μM [γ-³²P]ATP, 50 pmol 36-mer 5′-OH DNA, and 250 ng of the indicated HD-Pnk protein were incubated for 30 min at 22°C. The reactions were initiated by adding HD-Pnk and terminated by adding 10 μl of 80% formamide-100 mM EDTA, followed by heating for 5 min at 95°C. The products were analyzed by electrophoresis through a 15-cm 20% polyacrylamide gel containing 7 M urea in 45 mM Tris-borate and 1 mM EDTA. The radiolabeled oligonucleotide products were visualized by autoradiography of the gel and quantified by scanning the gel with a Typhoon FLA 9500 imaging apparatus. (C) Phosphatase reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 1 mM CuCl₂, 10 mM p-nitrophenylphosphate, and 2.5 μg of the indicated HD-Pnk protein were incubated at 30°C for 60 min. Reactions were quenched by adding 0.9 ml of 1 M Na₂CO₃, and the formation of p-nitrophenol was quantified by absorbance at 410 nm and interpolating the value to a p-nitrophenol standard curve. Product formation is plotted in bar graph format in panels B and C; each datum is the average of the results from three independent experiments ± the standard error of the mean (SEM).

FIG 3.

Metal cofactor specificity of the HD-Pnk kinase and phosphatase activities. (A) Kinase reaction mixtures (10 μl) containing 50 mM Tris-acetate (pH 6.5), 50 μM [γ-³²P]ATP, 50 pmol 36-mer 5′-OH DNA (depicted at the bottom), 100 ng HD-Pnk, and either no divalent cation (lane −) or 5 mM MgCl₂, MnCl₂, CaCl₂, CdCl₂, CoCl₂, CuCl₂, NiCl₂, or ZnCl₂, as specified, were incubated for 30 min at 22°C. The products were analyzed by urea-PAGE and visualized by autoradiography. (B) RNA 3′-phosphatase reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 50 nM ³²P-labeled 10-mer RNAp substrate (depicted at the bottom, with the labeled phosphate indicated by ●), 200 ng HD-Pnk, and either no divalent cation (lane −) or 5 mM MgCl₂, MnCl₂, CaCl₂, CdCl₂, CoCl₂, CuCl₂, FeCl₂, FeCl₃, NiCl₂, or ZnCl₂, as specified, were incubated for 30 min at 22°C. The products were analyzed by urea-PAGE and visualized by autoradiography. (C) RNA 2′,3′-cyclic-phosphodiesterase (CPDase) reaction mixtures (10 μl) containing 100 mM Tris-acetate (pH 6.0), 50 nM ³²P-labeled 10-mer RNA>p substrate (depicted at the bottom, with the labeled phosphate indicated by ●), 200 ng HD-Pnk, and either no divalent cation or 5 mM MgCl₂, MnCl₂, CaCl₂, CdCl₂, CoCl₂, CuCl₂, FeCl₂, FeCl₃, NiCl₂, or ZnCl₂, as specified, were incubated for 30 min at 22°C. The products were analyzed by urea-PAGE and visualized by autoradiography. The rightmost lane is a control reaction of the RNA>p substrate with the metal-independent CPDase of plant tRNA ligase AtRNL, which converts RNA>p to RNA_2′p. (D) Reaction mixtures (25 μl) containing 100 mM Tris-acetate (pH 6.0), 10 mM p-nitrophenylphosphate, 2.5 μg of HD-Pnk, and either no divalent cation (lane −) or 1 mM MgCl₂, MnCl₂, CaCl₂, CdCl₂, CoCl₂, CuCl₂, NiCl₂, or ZnCl₂, as specified, were incubated at 30°C for 60 min. The extents of p-nitrophenol formation are plotted in bar graph format; each datum is the average of the results from three independent experiments ± SEM.

FIG 4.

Size-exclusion chromatography of HD-Pnk. (A) HD-Pnk was gel filtered through a 25-ml Superdex 200 column in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM DTT, 2 mM EDTA, and 10% glycerol. The chromatographic profile (monitored by A₂₈₀ as a function of elution volume) is shown. The arrows specify the elution peaks and native sizes of a mixture of calibration standards: thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B₁₂ (1.4 kDa). (B) Kinase reaction mixtures (10 μl) containing 50 mM Tris-HCl (pH 8.5), 2 mM DTT, 5 mM MgCl₂, 50 μM 50 μM [γ-³²P]ATP, 50 pmol 36-mer 5′-OH DNA, and 2 μl of the even-numbered Superdex 200 fractions were incubated for 30 min at 22°C. The extents of DNA phosphorylation (in picomoles) are plotted on the y axis. (C) Phosphoesterase reactions mixtures (10 μl) containing 50 mM Tris-acetate (pH 6.0), 5 mM CoCl₂, 50 nM ³²P-labeled 10-mer RNA>p, and 2 μl of the even-numbered Superdex 200 fractions were incubated for 30 min at 22°C. The extents of conversion of RNA>p to RNA_OH (expressed as the percentage of total labeled RNA) are plotted on the y axis. Fraction 36 that has peak kinase and phosphoesterase activity (B and C) corresponds to the peak fraction in the elution profile shown in panel A.

Phosphoesterase activity of recombinant HD-Pnk.

We tested Deinococcus HD-Pnk for action as an RNA 3′-phosphomonoesterase and an RNA 2′,3′-cyclic-phosphodiesterase, these being two activities that were demonstrated for Runella HD-Pnk (19). To assay activity, we prepared 10-mer RNA substrates with either a 3′-phosphate (RNAp) or a 2′,3′-cyclic-phosphate (RNA>p) end and a single radiolabel between the 3′-terminal and penultimate nucleosides (Fig. 3B and C) (19). Because HD enzymes typically require transition metals as cofactors, we tested a broad range of metals at a 5 mM concentration for their ability to support an RNA 3′-end-healing reaction by Deinococcus HD-Pnk. We found by denaturing PAGE that cobalt and copper were uniquely effective in promoting an RNA 3′-phosphomonoesterase activity, evinced by conversion of the radiolabeled 10-mer RNAp substrate to a more slowly migrating RNA_OH product, with cobalt being the better cofactor (Fig. 3B).

The reaction of HD-Pnk with the RNA>p substrate in the presence of cobalt generated two products, an RNA with a monophosphate terminus (RNAp) that migrated faster than the input RNA>p, and an RNA_OH with no terminal phosphate that migrated more slowly (Fig. 3C). We surmise that HD-Pnk catalyzes sequential cyclic phosphodiesterase (CPDase) and phosphomonoesterase reactions at an RNA>p end. (The RNAp product of cyclic phosphodiester hydrolysis by HD-Pnk comigrates with the 2′-RNAp [RNA_2′p] product formed by reaction of the RNA>p substrate with the CPDase component of plant tRNA ligase AtRNL [27] [Fig. 3C]. However, because the urea-PAGE gel system does not separate RNA_2′p and 3′-RNAp [RNA_3′p] species, we cannot say from this analysis whether HD-Pnk hydrolyzes RNA>p to form exclusively a 3′ or 2′ phosphomonoester, or a mixture of both 2′ and 3′ phosphomonoesters.)

A salient finding was that a wider spectrum of metals sustained the RNA CPDase activity of HD-Pnk versus the RNA 3′-phosphomonoesterase activity (Fig. 3C versus B). To wit, manganese, cadmium, iron (ferrous and ferric), and zinc all supported detectable formation of RNAp but not of RNA_OH (Fig. 3C). Magnesium, calcium, and nickel were ineffective as cofactors for the HD-Pnk CPDase (Fig. 3C). Copper supported the formation of a mixture of RNAp and RNA_OH (Fig. 3C). Cobalt-dependent CPDase/phosphatase activity coeluted with the kinase activity and HD-Pnk protein during gel filtration, peaking in column fraction 36 (Fig. 4C).

In a prior study of Deinococcus HD-Pnk, Blasius et al. (18) were unable to assign a DNA 3′-phosphatase activity to the recombinant protein. The Runella HD-Pnk possesses DNA 3′-phosphatase activity but relies on copper (preferred), nickel, or cobalt as the divalent cation cofactor (19). It is conceivable that previous investigators did not use an effective metal cofactor for the HD hydrolase domain. Alternatively, the end-healing function of the HD component might be directed to a particular DNA substrate that was not interrogated. Here, we tested Deinococcus HD-Pnk for DNA 3′-phosphatase activity with the full spectrum of metals using the same 5′-³²P-labeled 10-mer 3′-DNA (DNA_3′p) substrate (pATCACGCTTCp) that was dephosphorylated by Runella HD-Pnk. We were unable to detect conversion of the DNA_3′p to DNA_OH by Deinococcus HD-Pnk (not shown), notwithstanding that a 10-mer RNA_3′p substrate of the same nucleobase sequence is dephosphorylated by Deinococcus HD-Pnk (Fig. 3B). We considered the possibility that Deinococcus HD-Pnk requires a specific nucleic acid configuration to function with DNA. To test this idea, we prepared a series of substrates in which an 18-mer 5′-³²P-labeled DNA_3′p strand (pCATATCCAATTGCGACCCp) was annealed to (i) a complementary cold 36-mer DNA oligonucleotide to form an 18-bp duplex with an 18-nucleotide 5′ tail (i.e., with a recessed 3′-phosphate end on the labeled strand), (ii) a complementary cold 17-mer DNA strand to form a 17-bp duplex with a 1-nucleotide 3′-phosphate overhang on the labeled strand, or (iii) a cold 17-nucleotide 5′-OH DNA oligonucleotide plus a 36-mer DNA oligonucleotide complementary to the labeled 18-mer and the cold 17-mer, to form a 36-mer duplex with a central 1-nucleotide gap with 3′-phosphate and 5′-OH gap termini. We found that HD-Pnk had no detectable DNA 3′-phosphatase activity on the 3′-recessed end, the 3′ overhang, the gapped duplex, or the 17-mer single-strand DNA substrates when assayed under conditions of 5-fold enzyme excess in the presence of either 1 mM cobalt or copper (not shown).

Finally, we queried whether Deinococcus HD-Pnk could react with the generic phosphomonoesterase substrate p-nitrophenylphosphate (pNPP), the hydrolysis of which liberates a chromogenic product p-nitrophenol that is easily quantified by its absorbance at 410 nm. We found that HD-Pnk has vigorous phosphatase activity with 10 mM pNPP in the presence of 1 mM copper as the metal cofactor (250 pmol p-nitrophenol formed per pmol HD-Pnk in a 60-min reaction), with other metals being ineffective (Fig. 3D). The generic phosphatase activity of HD-Pnk was effaced by the H81A-D82A mutation of the HD motif but was unimpeded by the K277A mutation in the Pnk P-loop motif (Fig. 2C). HD-Pnk did not generate detectable formation of p-nitrophenol when reacted with 10 mM bis-p-nitrophenylphosphate (a generic phosphodiesterase substrate) in the presence of any of the metals tested and shown in Fig. 3D (data not shown).

Neither the HD-Pnk gene nor the LIG–PARG–HD-Pnk operon is required for growth.

To examine whether HD-Pnk is required for growth, we deleted the HD-Pnk gene by the transformation of D. radiodurans with a knockout cassette composed of a kanamycin resistance gene (kanR) flanked by genomic DNA segments in the 5′ and 3′ directions of the HD-Pnk ORF (Fig. 5A). After multiple rounds of selection for kanamycin resistance, the intended HD-Pnk gene::kanR insertion and the absence of an intact HD-Pnk gene were confirmed by PCR of genomic DNA with diagnostic primers (Fig. 5B). Western blot analysis with anti-HD-Pnk antibody raised against recombinant HD-Pnk and affinity-purified verified its reactivity with recombinant HD-Pnk and the detection of a polypeptide of the same size in a whole-cell extract of wild-type D. radiodurans but not in the ΔHD-Pnk mutant strain (Fig. 5D). The antibody also recognized an ∼33-kDa polypeptide (denoted by the asterisk in Fig. 5D) that was present in wild-type and ΔHD-Pnk mutant cell extracts.

FIG 5.

Construction of ΔHD-Pnk and ΔLIG–PARG–HD-Pnk mutant strains. (A) Diagram of the LIG–PARG–HD-Pnk gene cluster in wild-type D. radiodurans (WT) and after homologous recombination with the kanR-marked deletion cassettes to singly delete the HD-Pnk gene (ΔHD-Pnk) or to delete the entire three-gene cluster (ΔLIG–PARG–HD-Pnk). The cooriented ORFs encoding HD-Pnk, PARG, and LIG are depicted as labeled arrows, as is the kanR cassette. The positions of the diagnostic PCR primers used for genotyping are shown. (B) PCR confirmation of the ΔHD-Pnk mutant using the diagnostic primers shown. The PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The positions and sizes (in kilobase pairs) of linear DNA size markers are indicated on the left. The expected sizes of the PCR products from WT and ΔHD-Pnk strains are 2,902 bp and 2,626 bp, respectively. (C) PCR confirmation of the ΔLIG–PARG–HD-Pnk strain using two distinct primer pairs, as shown. The positions and sizes (in kilobase pairs) of linear DNA size markers are indicated on the left. The expected size of the ΔLIG–PARG–HD-Pnk PCR product on the left side is 1,517 bp. The expected size of the WT PCR product in the right panel is 2,902 bp. (D) Western blot using an anti-HD-Pnk antibody after SDS-PAGE of whole-cell extracts of equivalent aliquots (A₆₀₀) of exponentially growing wild-type, ΔHD-Pnk mutant, and ΔLIG–PARG–HD-Pnk mutant cells and aliquots (1, 2, 5, and 10 ng) of purified recombinant HD-Pnk. The positions and sizes (in kilodaltons) of marker polypeptides are indicated on the left. The HD-Pnk polypeptide is denoted by an arrow at the right. A 33-kDa polypeptide that is recognized by the antibody is denoted by an asterisk at the left.

We proceeded to construct a knockout strain in which the entire LIG–PARG–HD-Pnk operon was deleted and replaced with a kanR marker (Fig. 5A). The genotype of the ΔLIG–PARG–HD-Pnk mutant strain was confirmed by PCR of genomic DNA with diagnostic primers (Fig. 5C), and the absence of the HD-Pnk protein was affirmed by Western blotting (Fig. 5D). There was no difference in the growth rates of wild-type, ΔHD-Pnk mutant, and ΔLIG–PARG–HD-Pnk mutant strains in tryptone-glucose-yeast (TGY) medium at 30°C (not shown).

Deletion of the HD-Pnk gene sensitizes D. radiodurans to gamma irradiation.

The survival of wild-type and ΔHD-Pnk mutant strains was assessed after exposure to increasing doses of gamma radiation from a ¹³⁷Cs source. The survival of wild-type D. radiodurans was unaffected by 3 and 6 kGy of IR exposure but declined steadily thereafter as the dosage was increased to 9 kGy (12% survival) and 12 kGy (0.86% survival) (Fig. 6A). In contrast, the ΔHD-Pnk mutant strain was sensitized to IR doses exceeding 3 kGy, with survival rates of 7.7% at 6 kGy and 0.16% at 9 kGy (Fig. 6A). No ΔHD-Pnk mutant survivors were detected after exposure to 12 kGy of IR (detection limit in this experiment being 0.01% survival). Compared to the wild-type strain, the ΔHD-Pnk mutant was 11-fold and 80-fold more sensitive to killing by 6 kGy and 9 kGy of gamma radiation, respectively. These results implicate HD-Pnk as an agent of nucleic acid repair in response to IR damage.

FIG 6.

Deletion of the HD-Pnk gene sensitizes D. radiodurans to gamma irradiation. (A) IR sensitivity. Wild-type, ΔHD-Pnk mutant, and ΔLIG–PARG–HD-Pnk mutant cells were irradiated on ice in 500-μl volumes using a ¹³⁷Cs gamma-ray source. Aliquots were removed after receiving the indicated doses, and serial dilutions were plated on TGY agar. The survival of each strain, normalized to a mock-irradiated control, is plotted on a log-scale y axis as a function of IR dose. Each datum is the average of the results from three or four separate experiments ± SEM. (B) Genome reassembly during recovery from IR. Wild-type and ΔHD-Pnk mutant cells were exposed to a 9-kGy dose of IR. Aliquots of cells were removed 0, 3, 6, 9, and 12 h during the post-IR outgrowth and processed for PFGE of proteinase K-digested agarose plugs, as described in Materials and Methods. Mock-irradiated control cells were analyzed in the lanes labeled −IR. A negative image of the ethidium bromide-stained gel is shown. Phage λ DNA concatemers were analyzed in parallel (in λ lanes), where the positions and sizes (in kilobases) of selected λ DNAs in the ladder are indicated on the left. Three of the genomic replicons, a 46-kbp plasmid, a 177-kbp megaplasmid, and a 414-kbp secondary chromosome, are denoted by dots next to WT, lane −IR. (C) HD-Pnk is depleted early during recovery from IR and replenished at later stages. Wild-type D. radiodurans cells exposed to a 9-kGy dose of IR were diluted to an A₆₀₀ of 0.4 in TGY medium and then incubated at 30°C with constant shaking. Aliquots were removed at the times specified during post-IR recovery, and whole-cell extracts were analyzed by SDS-PAGE and immunoblotting with anti-HD-Pnk antibody (top) and anti-GlnA antibody (bottom). The positions and sizes (in kilodaltons) of marker polypeptides are indicated on the left on the top.

The IR sensitivity of the ΔLIG–PARG–HD-Pnk mutant strain in which the entire operon was deleted was virtually identical to that of the ΔHD-Pnk mutant strain (Fig. 6A). This result is significant in light of the report by Kota et al. (28) that deletion of the LIG ORF by replacement with a selectable marker sensitized D. radiodurans to a 10-kGy IR exposure, but rescue of the IR sensitivity could not be achieved by expressing the ligase polypeptide alone. Rather, the recovery of IR resistance required expression of the entire three-gene operon (28). It was not clear from their data which of the three proteins of the operon were relevant to the IR phenotype. Our data prompt us to suggest that a polar effect of the marker insertion into the LIG gene that affected the downstream HD-Pnk gene was responsible for the IR sensitivity observed by Kota et al. (28).

HD-Pnk is depleted early during recovery from IR and replenished at late stages.

The fate of the HD-Pnk protein in wild-type D. radiodurans cells immediately after exposure to 9 kGy of IR and during post-IR recovery upon reinoculation into TGY medium was analyzed by Western blotting (Fig. 6C). HD-Pnk was present immediately after 9 kGy of exposure (time zero) but was depleted after 3 h of post-IR recovery and remained depleted at 6 h and 9 h. HD-Pnk began to increase at 12 h and was restored by 15 h to the same level seen at time zero (Fig. 6C). This early depletion and late repletion of HD-Pnk were not reflective of generalized protein turnover post-IR, insofar as the level of the 33-kDa polypeptide recognized by the antibody remained fairly constant during the same period, as did the level of the GlnA protein that was probed by immunoblotting with anti-GlnA antibody (Fig. 6C). These results suggest that HD-Pnk is turned over early during recovery from gamma irradiation, conceivably because it suffers IR-induced oxidative damage. The replenishment of HD-Pnk levels in the late stages of recovery has implications for when HD-Pnk might function in radiation resistance.

Genome reassembly after 9 kGy of gamma irradiation.

Pulsed-field gel electrophoresis (PFGE) was employed to monitor genome reassembly in wild-type and ΔHD-Pnk mutant cells after exposure to 9 kGy of gamma radiation. Equivalent aliquots (by A₆₀₀) of cells immobilized in agarose plugs were subjected to PFGE in parallel with a bacteriophage λ DNA concatemer ladder (Fig. 6B). The four DNA replicons that comprise the Deinococcus genome (29) were evident in the mock-irradiated samples, these being a 46-kbp plasmid, a 177-kbp megaplasmid, and a 414-kbp secondary chromosome (Fig. 6B, wild type [WT], lane −IR; denoted by dots) and a 2.65-Mbp main chromosome. Exposure to 9 kGy of IR shattered the genome into DNA fragments of <49 kbp (Fig. 6B, time zero). Genome reassembly, manifested as the restoration of the replicons, was evident in wild-type cells at 9 h post-IR, following a 6-h-long “early” phase devoted to DNA resection and synthesis-dependent strand annealing (5, 30) in which the shattered fragments are either unchanged (Fig. 6B, 3 h) or diminished (Fig. 6B, 6 h). By 12 h post-IR, wild-type cells had progressed further in genome reconstitution, as gauged by the increased abundance of the main chromosome and the decrement in the smallest shattered fragments. In the ΔHD-Pnk mutant strain, the DNA profile during the 12-h post-IR period was similar to that of wild-type cells, with respect to the 6-h early phase and the reappearance of the main chromosome at 9 to 12 h. These results, together with the time or replenishment of HD-Pnk during post-IR recovery, suggest that HD-Pnk participates in repair transactions that come into play late, i.e., after reassembly of the genomic replicons from heterogeneous DNA fragments with double-strand breaks.

Introduction of HD-Pnk active-site mutations by allelic exchange.

Because HD-Pnk is a bifunctional enzyme, any phenotype observed for ΔHD-Pnk mutant cells could reflect the absence of its 3′-phosphoesterase or 5′-kinase functions, or both. Alternatively, the HD-Pnk protein might play a role independent of its catalytic activities, e.g., as a structural component of a repair complex. To address this issue, we reintroduced HD-Pnk alleles encoding wild-type HD-Pnk protein, the kinase-dead/phosphatase-active mutant K277A, or the kinase-active/phosphatase-dead mutant H81A-D82A into the genomic LIG–PARG–HD-Pnk operon. To achieve allelic exchange, we constructed a DNA clone derived from the native LIG–PARG–HD-Pnk operon that includes the 3′ half of the PARG gene and the entire HD-Pnk gene (either wild type, H81A-D82A, or K227A), plus a cooriented downstream aadA gene conferring streptomycin resistance and a 3′-flanking segment corresponding to genomic DNA downstream of the LIG–PARG–HD-Pnk operon. This DNA cassette was transfected into the ΔHD-Pnk mutant strain, wherein the marked cassette could recombine, directed by the flanking homologous sequences, into the ΔHD-Pnk::kanR locus so as to restore the original intact three-ORF operon (expressing wild-type or mutant HD-Pnk) (Fig. 7A). The correct insertion was selected by gain of streptomycin resistance and then screening streptomycin-resistant (Strep^r) transformants for loss of kanamycin resistance. Allelic replacement was confirmed by diagnostic PCR of genomic DNA (Fig. 7B). Finally, the HD-Pnk ORFs of the HD-Pnk gene–aadA strains were sequenced to verify the wild-type and mutant alleles, respectively. Testing of the HD-Pnk gene–aadA strains by Western blotting with anti-HD-Pnk antibody affirmed that they regained expression of the HD-Pnk protein (vis à vis the ΔHD-Pnk starting strain) at levels similar to that of wild-type D. radiodurans (Fig. 7C).

FIG 7.

HD-Pnk active-site mutations sensitize Deinococcus to mitomycin C. (A) Introduction of HD-Pnk active-site mutations by allelic exchange. Diagram of the LIG–PARG–HD-Pnk operon after homologous recombination of the ΔHD-Pnk::kanR locus with one of three HD-Pnk gene–aadA knock-in cassettes (with HD-Pnk wild-type, H81A-D82A, or K277A alleles) that confers streptomycin resistance. The positions of the diagnostic PCR primers used for genotyping are shown. The primers are located outside the margins of the 250-bp flanking homology sequences in the knock-in cassette. (B) Confirmation of the correct knock-in loci by PCR amplification of genomic DNA isolated from the indicated strains using the primers shown in panel A. The positions and sizes (in kilobase pairs) of linear DNA size markers are indicated on the left. The expected sizes of the PCR products from the WT, ΔHD-Pnk mutant, and HD-Pnk gene–aadA mutant strains are 2,902 bp, 2,626 bp, and 4,029 bp, respectively. (C) Western blotting of lysates of equivalent aliquots of cells (as measured by A₆₀₀) with anti-HD-Pnk antibody. The positions and sizes (in kilodaltons) of marker polypeptides are indicated on the left. HD-Pnk is indicated by an arrow at right. (D) The HD-Pnk gene–aadA strains (with WT, H81A-D82A, or K277A HD-Pnk alleles, as indicated) and the ΔHD-Pnk mutant strain were incubated at 30°C for 10 min with mitomycin C. Survival (normalized to the mock-treated control) is plotted as a function of mitomycin C concentration. Each datum is the average of the results from three separate experiments ± SEM.

HD-Pnk active-site mutations sensitize Deinococcus to killing by mitomycin C.

In light of the findings of Kota et al. (28) that their ΔLIG mutant strain (in which the LIG–PARG–HD-Pnk operon was disrupted) was sensitized to killing by mitomycin C (which generates DNA interstrand cross-links [32]), we turned to mitomycin C as a potentially more user-friendly probe of HD-Pnk function. In lieu of the lengthy exposures to a ¹³⁷Cs source required for performance of IR sensitivity experiments, we simply exposed logarithmically growing D. radiodurans cultures to various concentrations of mitomycin C for 10 min, harvested and washed the cells to remove the mitomycin C, and plated serial dilutions on agar to gauge survival, normalized to a control culture of the same strain that had not been exposed to mitomycin C. The mitomycin C sensitivity profiles of the wild-type, H81A-D82A mutant, and K227A HD-Pnk gene–aadA mutant strains are shown in Fig. 7D. The salient findings were that active-site mutations that effaced either the kinase or the phosphoesterase functions of HD-Pnk rendered cells ∼20-fold more sensitive to 20 μg/ml mitomycin and ∼50-fold more sensitive to 30 μg/ml mitomycin compared to the isogenic wild-type HD-Pnk gene–aadA strain (Fig. 7D). The mitomycin C sensitivity profiles of the active-site mutants paralleled that of the ΔHD-Pnk null strain (Fig. 7D). We surmise from these results that both catalytic activities of HD-Pnk are pertinent to the repair of nucleic acid damage caused by mitomycin C.

DISCUSSION

In this study, we interrogated biochemically and genetically the activities of Deinococcus HD-Pnk, a member of a family of bifunctional nucleic acid end-healing enzymes composed of an N-terminal HD phosphoesterase domain and a C-terminal P-loop phosphotransferase domain (19). Recombinant HD-Pnk is a magnesium-dependent polynucleotide kinase that relies on the conserved lysine that defines the P-loop motif. HD-Pnk also catalyzes metal-dependent 2′,3′-CPDase and 3′-phosphomonoesterase reactions at polynucleotide ends. HD-Pnk is not a nuclease (i.e., 3′-5′-phosphodiesterase), as gauged by the stability of the 3′-³²P-labeled products of the HD-Pnk 3′-healing reactions. HD-Pnk also has phosphomonoesterase activity on the generic substrate p-nitrophenylphosphate. The phosphatase activity relies on the eponymous His and Asp side chains of the HD motif. The kinase and phosphoesterase activities are not interdependent, insofar as a crippling mutation of the kinase active site does not affect the phosphatase activity and vice versa. In these respects, Deinococcus HD-Pnk resembles the Runella slithyformis HD-Pnk enzyme (19).

Deinococcus HD-Pnk displays distinctive metal cofactor specificities for its phosphoesterase activities. The hydrolysis of p-nitrophenylphosphate is exclusively reliant on copper (Fig. 3D); this is a property shared with Runella HD-Pnk (A. Munir, unpublished data). The RNA 3′-phosphomonoesterase activity is supported by cobalt (preferred) and copper but not by nickel. In contrast, nickel is an effective cofactor for the RNA 3′-phosphomonoesterase of Runella HD-Pnk (19). Manganese, cadmium, and cobalt were the preferred cofactors for the 2′,3′-CPDase activity of Deinococcus HD-Pnk (Fig. 3C). In the case of Runella HD-Pnk, 2′,3′-CPDase activity was optimal with nickel, manganese, cobalt, and copper (19).

Previously, Blasius et al. (18) demonstrated 5′-OH DNA kinase activity of Deinococcus HD-Pnk in the presence of manganese but were unable to assign a DNA 3′-phosphatase activity to the recombinant protein. Blondal et al. (31) characterized an HD-Pnk enzyme encoded by the thermophilic bacteriophage RM378. They demonstrated a capacity of the RM378 enzyme to release phosphate from a cyclic mononucleotide substrate 2′,3′-cAMP in the presence of manganese, but they were unable to detect DNA 3′-phosphatase activity (31). The Runella HD-Pnk possesses DNA 3′-phosphatase activity but relies on copper (preferred), nickel, or cobalt as the divalent cation cofactor (19). It is conceivable that the failure of previous investigators to assign a polynucleotide 3′-end-healing function to their HD-Pnk protein stems from the use of an ineffective metal cofactor for the HD hydrolase domain. Alternatively, the end-healing function of the HD component might be directed to a particular nucleic acid substrate that was not interrogated. With respect to that point, we were unable to detect a DNA 3′-phosphatase activity of Deinococcus HD-Pnk with a variety of DNA strand configurations using metal cofactors that supported its hydrolysis of RNA 3′-phosphoesters (or p-nitrophenylphosphate). It would appear that the 3′-end-healing phosphoesterase activities of Deinococcus HD-Pnk are RNA-end specific, conceivably because the ribose 2′-OH plays a role in end recognition or catalysis.

We show here that Deinococcus HD-Pnk is present in logarithmically growing bacteria yet is inessential for growth under laboratory conditions. Knockout of the HD-Pnk gene sensitizes Deinococcus to killing by ≥6 kGy of gamma irradiation, a finding that provides the first evidence that HD-Pnk plays a role in nucleic acid repair. The dynamics of HD-Pnk during post-IR recovery are quite striking, insofar as the HD-Pnk protein is depleted during the early phases when the shattered genomic fragments undergo resection, synthesis-dependent strand annealing (SDSA), and homologous recombination to begin reassembly of the four genomic replicons. This early disappearance runs counter to the expectation that putative 3′ DNA end-healing activity of HD-Pnk might be necessary at early times to convert the dirty 3′ ends resulting from radiation damage into clean 3′-OH ends that serve as primers for the SDSA phase of genome reconstitution. Yet, the early disappearance is in accord with the observation that the ΔHD-Pnk null mutant can convert the shattered fragments into larger products of replicon size. Therefore, we conclude that Deinococcus relies on enzymatic agents other than HD-Pnk (e.g., a 3′-nuclease or a distinct 3′-phosphoesterase) to generate the clean 3′-OH ends at radiation-inflicted breaks that are needed for SDSA. Our results do not distinguish whether (i) HD-Pnk can perform this function in vivo (notwithstanding its lack of observable DNA 3′-phosphatase activity in vitro) but is genetically redundant to other 3′ end-processing enzymes, or (ii) HD-Pnk is not adept at healing DNA 3′ ends damaged by IR.

HD-Pnk protein starts to reaccumulate at 9 h post-IR and is fully replete at 15 h. The depletion and repletion of HD-Pnk during postradiation recovery echo what was observed for the DraRnl protein after a 10-kGy dose of IR (10). The restoration of HD-Pnk protein occurs at a time when reassembly of the Deinococcus genome is far advanced. Indeed, the observation that genome reassembly, as gauged by pulsed-field gel electrophoresis, is not prevented in ΔHD-Pnk mutant cells suggests that the IR-sensitivity accompanying loss of HD-Pnk could result from a defect in the repair of residual DNA lesions in the reassembled replicons.

We envision a function for HD-Pnk in the repair of residual single-strand gaps in the DNA duplex, whereby HD-Pnk heals ends in preparation for the action of a gap repair polymerase and a nick-sealing ligase. The in vitro activity of the HD-Pnk phosphoesterase on ribonucleotide 3′ ends raises the prospect that ribonucleotides are introduced into DNA during the repair of IR damage to the Deinococcus genome and HD-Pnk is adapted to healing breaks at the sites of ribonucleotide incorporation.

We report here that HD-Pnk also plays a role in the resistance of Deinococcus to killing by transient exposure to 20 to 30 μg/ml mitomycin C. Mitomycin C is a potent cytotoxic antibiotic that upon reductive activation forms monofunctional adducts and interstrand covalent cross-links to the guanine N-2 positions at CG dinucleotide steps in DNA (32). By constructing allelic replacements with HD-Pnk gene mutants that are uniquely crippled in their kinase or phosphoesterase activity, we provide evidence that both end-healing reactions are needed to carry out the functions of HD-Pnk in recovery from mitomycin C damage. Thus, HD-Pnk joins a variety of other Deinococcus DNA repair proteins (e.g., proteins implicated in nucleotide excision repair and homologous recombination), mutants of which are sensitized to killing by mitomycin C (reviewed in reference 5).

To our knowledge, the present study is the first to implicate a bacterial polynucleotide kinase-phosphatase in resistance to treatments that damage DNA. Because the standard laboratory strains of E. coli lack a polynucleotide kinase-phosphatase enzyme, the question would not have been addressed previously in that model system. However, it is the case that pathogenic strains E. coli UTI89 and O127:H6 do have an HD-Pnk gene in the context of a LIG–PARG–HD-Pnk operon (19); the function of these genes has not been examined. In contrast, investigations of the bifunctional DNA 5′-polynucleotide kinase/3′-phosphatase (PNKP) present in eukarya, which consists of a proximal phosphatase domain of the DXDXT acyl-phosphatase superfamily and a distal P-loop kinase domain (12), show that PNKP plays a key role in the repair of DNA breaks inflicted by gamma radiation, topoisomerase IB poisons, or hydrogen peroxide (33, 34).

In closing, it is worth noting that the biochemical requirements of both HD-Pnk and DraRnl for 3′-terminal ribonucleotides in order to execute their respective 3′-phosphoesterase and 3′-OH/5′-PO₄ ligation activities raise the possibility that these enzymes participate in the repair of RNA damage elicited by exposure of Deinococcus spp. to high-dose IR or toxic concentrations of mitomycin C. Along the same lines, Han et al. (35) recently reported that another putative Deinococcus RNA end-processing enzyme, DR_2239, plays an important role in resistance to nucleic acid damage. The DR_2239 gene, which is strongly transcriptionally upregulated during recovery from IR (25, 26), encodes a member of the 2H phosphoesterase enzyme superfamily (36). Disruption of the DR_2239 gene renders Deinococcus 5-fold more sensitive to 10 kGy of IR exposure but 2,000-fold more sensitive to treatment with 40 μg/ml mitomycin C vis à vis the wild-type strain (35). The homologous protein in E. coli has been characterized structurally and biochemically as an RNA 2′,3′-cyclic-phosphodiesterase that hydrolyzes the cyclic-phosphate terminus to form an RNA 2′-phosphomonoester (37). Whereas RNA damage infliction and repair in Deinococcus spp. are virtually a tabula rasa, there is an emerging appreciation from studies in other systems that RNAs are potentially meaningful targets for damage by reactive oxygen species and ostensibly DNA-damaging drugs (38–42).

MATERIALS AND METHODS

Recombinant Deinococcus HD-Pnk.

Plasmid pET28B-His₁₀-Smt3-HD-Pnk encodes the 413-aa HD-Pnk polypeptide fused to an N-terminal His₁₀-Smt3 tag. The expression plasmid was transformed into Escherichia coli BL21(DE3). Cultures (8 of 1 liter) derived from single kanamycin-resistant transformants were grown at 37°C in Terrific broth medium containing 30 μg/ml kanamycin until the A₆₀₀ reached 0.6, at which time the cultures were adjusted to 0.1 mM isopropyl-β-d-thiogalactoside (IPTG) and 2% (vol/vol) ethanol and then incubated for 16 h at 17°C with continuous shaking. Cells were harvested by centrifugation, resuspended in 200 ml of lysis buffer (50 mM Tris-HCl [pH 8.0], 300 mM NaCl, 20 mM imidazole, 10% sucrose), and stored at −80°C. All subsequent procedures were performed at 4°C. The thawed cell suspension was supplemented with lysozyme (1 mg/ml final concentration), four Roche cOmplete Mini protease inhibitor tablets (EDTA free), and Triton X-100 (0.1% final concentration). The lysate was sonicated to reduce viscosity, and insoluble material was removed by centrifugation at 14,000 rpm for 45 min. The supernatant was applied to an 8-ml column of His₆₀ Ni Superflow resin (Clontech) that had been equilibrated in lysis buffer. The column was washed with buffer A (50 mM Tris-HCl [pH 8.0], 250 mM NaCl, 10% glycerol) containing 20 mM imidazole, and bound material was eluted stepwise with buffer A containing 50, 100, 200, and 500 mM imidazole. The elution profile of the His₁₀-Smt3-HD-Pnk polypeptide was monitored by SDS-PAGE. The 200 mM and 500 mM imidazole fractions containing His₁₀-Smt3-HD-Pnk were pooled and supplemented with Smt3-protease Ulp1 (1:100 ratio of Ulp1 to His₁₀-Smt3-HD-Pnk). The protein was dialyzed overnight against buffer A. The dialysate containing the cleaved His₁₀-Smt3 tag and tag-free HD-Pnk was applied to a 4-ml His₆₀ column equilibrated in buffer A with 20 mM imidazole, and tag-free HD-Pnk was recovered in the flowthrough. HD-Pnk protein concentrations were determined with the Bio-Rad dye-binding reagent using bovine serum albumin as a standard.

Gel filtration.

The tag-free wild-type HD-Pnk preparation was concentrated by centrifugal ultrafiltration (10,000 molecular weight cutoff; Amicon) and applied to a 25-ml Superdex 200 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM dithiothreitol (DTT), 2 mM EDTA, and 10% glycerol at a flow rate of 1 ml/min while collecting 0.5-ml fractions. The gel filtration column was calibrated by applying a mixture of size standards containing thyroglobulin, γ-globulin, ovalbumin, myoglobin, and vitamin B₁₂.

HD-Pnk mutants.

The alanine K277A and H81A-D82A mutations were introduced into the pET28B-His₁₀-Smt3-HD-Pnk expression vector, and the HD-Pnk ORFs were sequenced to confirm that no unwanted coding changes were acquired. The K277A and H81A-D82A proteins were purified from 3-liter cultures of IPTG-induced Escherichia coli BL21(DE3), in parallel with wild-type HD-Pnk, via sequential Ni-affinity, tag cleavage, and tag removal steps, as described above. SDS-PAGE showed that the 45-kDa HD-Pnk polypeptide was the major species in the enzyme preparations; an unidentified minor ∼70-kDa polypeptide was also present (Fig. 2A).

3′-Phosphoesterase assays.

We prepared 10-mer RNAs with either a 3′-phosphate (RNAp) or a 2′,3′-cyclic-phosphate (RNA>p) end and a single radiolabel between the 3′-terminal and penultimate nucleosides, as described previously (19). RNA phosphoesterase reaction mixtures were constituted as described in the figure legends. The reactions were quenched by adding an equal volume of 80% formamide and 100 mM EDTA and heating for 5 min at 95°C. The products were analyzed by electrophoresis at 50-W constant power through a 40-cm 20% polyacrylamide gel containing 8 M urea in 45 mM Tris-borate and 1.2 mM EDTA. The ³²P-labeled RNAs were visualized by autoradiography of the gel and, where specified, quantified by scanning the gel with a Typhoon FLA 9500 imager.

Growth of D. radiodurans.

D. radiodurans R1 strains were grown in 1× TGY broth (0.5% tryptone, 0.1% glucose, 0.15% yeast extract) or agar (1.5%) at 30°C. Liquid cultures were incubated on a platform shaker at 250 rpm, and growth was measured by monitoring the absorbance of the cultures at 600 nm.

Construction of D. radiodurans HD-Pnk mutant and ΔLIG–PARG–HD-Pnk mutant strains.

To replace the HD-Pnk gene with a kanamycin resistance marker (kanR), a knockout cassette consisting of 250 bp of genomic DNA upstream of the HD-Pnk gene, a pkat-kanR kanamycin resistance gene driven by the constitutive promoter of the Deinococcus katA gene, and 250 bp of genomic DNA downstream of the HD-Pnk gene was synthesized by a commercial source (GenScript), inserted into pUC19, and sequenced. To replace the LIG–PARG–HD-Pnk operon with pkat-kanR, a knockout cassette consisting of 901 bp of genomic DNA upstream of the LIG gene, the pkat-kanR kanamycin resistance marker, and 901 bp of DNA downstream of the HD-Pnk gene was synthesized (Genewiz), inserted into pUC57-Amp, and sequenced. The pUC19-HD-Pnk-KO and pUC57-OPERON-KO plasmids (which cannot replicate autonomously in D. radiodurans) were transfected into CaCl₂ competent D. radiodurans. Transformants were selected on TGY agar containing 10 μg/ml kanamycin. Several rounds of kanamycin selection, cycling between liquid and solid TGY medium, were carried out. Correct integration of the kanR marker in lieu of the HD-Pnk or LIG–PARG–HD-Pnk gene was confirmed by PCR with diagnostic primers (Fig. 5B and C).

Allelic replacement of ΔHD-Pnk locus with HD-Pnk gene–pkat-aadA.

To replace the ΔHD-Pnk locus with HD-Pnk gene–pkat-aadA, five DNA parts consisting of (i) 220 bp of genomic PARG DNA immediately upstream of the HD-Pnk gene, (ii) the complete HD-Pnk gene (encoding wild-type, kinase-dead, or phosphoesterase-dead enzymes), (iii) the pkat-aadA streptomycin resistance cassette from pTNK103 (26), (iv) 250 bp of DNA downstream of the HD-Pnk gene, and (v) BamHI-linearized pUC19 vector were assembled in a one-pot Gibson PCR (NEB) incubated for 1 h at 50°C in order to generate the knock-in pUC-PARG–HD-Pnk–aadA-KI plasmids with wild-type or mutated HD-Pnk alleles. The 2,843-bp HD-Pnk gene–pkat-aadA fragment was generated by PCR amplification using overlapping primers suitable for Gibson PCR. The pUC19 vector was linearized with BamHI, treated with Antarctic phosphatase, and gel purified prior to use in the Gibson assembly. CaCl₂ competent D. radiodurans ΔHD-Pnk gene::kanR cells were transfected with the pUC-PARG–HD-Pnk–aadA-KI plasmids. Transformants were selected on TGY agar medium containing 5 μg/ml streptomycin. Three rounds of streptomycin selection, cycling between liquid and solid TGY media, were carried out at escalating doses of streptomycin ranging from 5 μg/ml in the first round to 20 μg/ml in the final round. Correct integration was confirmed by PCR of genomic DNA (Fig. 7B). The genotypes at the reintegrated HD-Pnk loci were confirmed by PCR amplification of the ORF and sequencing to affirm the wild-type allele and the presence of the desired alanine mutations in the kinase or phosphoesterase domains.

Immunodetection of HD-Pnk in D. radiodurans whole-cell extracts.

Purified recombinant HD-Pnk was used to generate rabbit polyclonal antibodies according to the Pocono Hill Rabbit Farm (Canadensis, PA) 91-day protocol. Anti-HD-Pnk was affinity purified from serum by adsorption to covalently coupled HD-Pnk using the Pierce Gentle Ag/Ab binding and elution buffer kit. Purified antibody was dialyzed against phosphate-buffered saline (PBS), aliquoted, and stored at −80°C. For Western analysis, cells were resuspended in 10 mM sodium phosphate (pH 7.5), mixed with 4× SDS loading dye, and lysed by heating for 5 min at 95°C. Lysates were spun for 1 min at 14,000 rpm in a microcentrifuge, and the supernatants were analyzed by 12% SDS-PAGE gels. Polypeptides were electrophoretically transferred from the gel to a Hybond Protran nitrocellulose membrane (GE Healthcare). Membranes were preincubated with blocking buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween 20, 5% powdered milk). HD-Pnk was detected using the anti-HD-Pnk antibody at a dilution of 1:1,000 in blocking buffer. The secondary antibody, anti-rabbit IgG–horseradish peroxidase (IgG-HRP) conjugate, was obtained from Cell Signaling. GlnA was detected using chicken anti-GlnA primary antibody (Agrisera) at a dilution of 1:5,000 and a goat anti-chicken IgY–HRP secondary antibody (Santa Cruz Biotechnology). Both secondary HRP conjugates were incubated with Pierce ECL Western blotting substrate for visualization.

Gamma irradiation.

Cells were grown to mid-log phase (A₆₀₀, ∼0.4), washed in 10 mM sodium phosphate, and then concentrated 100-fold in the same buffer prior to gamma irradiation. The concentrated cell suspensions were exposed on ice in 500-μl volumes to a ¹³⁷Cs gamma-ray source (J. L. Shepherd Mark I) at a dose rate of 9.4 Gy/min. (The ice was replenished several times during the radiation exposure.) Aliquots were removed, diluted, and plated on TGY agar. Survivors were scored by counting colonies on TGY agar after growth at 30°C for 3 to 4 days.

Gamma-radiation outgrowth in liquid cultures.

Cells irradiated with a 9-kGy dose were diluted in TGY medium to an A₆₀₀ of 0.4 and then incubated at 30°C with constant shaking at 250 rpm. Aliquots were withdrawn at various times during the postirradiation recovery period, centrifuged at 5,000 × g, resuspended in 10 mM sodium phosphate (pH 7.5), and then processed for immunoblotting.

Pulsed-field gel electrophoresis.

Cells irradiated with a 9-kGy dose were diluted 100-fold in TGY medium and incubated at 30°C with constant shaking. At various times during the postirradiation outgrowth, aliquots were removed to prepare DNA plugs, as described previously (43). Proteinase K-digested agarose plugs were subjected to pulsed-field gel electrophoresis for 22 h at 12°C using the Chef-DR III electrophoresis system (Bio-Rad). The electrophoresis was carried out at 6 V/cm, with a linear pulse of 10 to 60 s and a switching angle of 120°. Bacteriophage λ cI857-Sam7 DNA concatemers (Bio-Rad) were analyzed in parallel to provide size standards.

Mitomycin C sensitivity.

Mid-log-phase 0.5-ml cultures (A₆₀₀, ∼0.4) were supplemented with 32 μl of mitomycin C in 50% dimethyl sulfoxide (DMSO) to attain a final concentration of 10, 20, or 30 μg/ml mitomycin C. Control cultures received 32 μl of 50% DMSO without drug. After a 10-min incubation at 30°C, cells were harvested by centrifugation, washed in 10 mM sodium phosphate (pH 7.5), and then resuspended in the same buffer. Serial 10-fold dilutions were plated on TGY agar, and survivors were scored by counting colonies after growth at 30°C for 3 to 4 days.