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PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans
BBRC Biochemical and Biophysical Research Communications 306 (2003) 354–360 www.elsevier.com/locate/ybbrc
PprI: a general switch responsible for extreme radioresistance of Deinococcus radioduransq Yuejin Hua,a,b,* Issay Narumi,b Guanjun Gao,a Bing Tian,a Katsuya Satoh,b Shigeru Kitayama,b and Binghui Shenc b
a Institute of Nuclear-Agricultural Science, Zhejiang University, Hangzhou 310029, China Biotechnology Laboratory, Takasaki Radiochemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki 370-1292, Japan c Division of Molecular Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010-3000, USA
Received 5 May 2003
Abstract Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress. Ó 2003 Elsevier Science (USA). All rights reserved.
The extremely radioresistant bacterium, Deinococcus radiodurans, is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet radiation, and desiccation [1,2]. Its unprecedented ability to avoid DNA damage in the presence of heavy irradiation has made it a critical model for pursuing genes involved in maintaining DNA integrity and stability. For decades, scientists have been attempting to delineate the mechanisms by which this bacterium overcomes these extreme levels of DNA damage-inducing agents. The bacteriumÕs phenomenal radioresistance was found to derive from its ability to repair hundreds of double-strand DNA breaks [3,4]. D. radiodurans is able to reconstruct a functional genome from chromosomal fragments, q
Abbreviations: PprI, inducer of pleiotropic proteins promoting DNA repair; PprA, pleiotropic protein promoting DNA repair A; RecA, recombinase A. * Corresponding author. Fax: +86-571-8697-1703. E-mail address: [email protected] (Y. Hua).
whereas the genomes of most organisms are irreversibly shattered by the effects of high levels of ionizing radiation [5–9]. This process was thought to occur mainly via homologous recombination requiring RecA (recombinase A) function [10–13], although DNA repair via other RecA-independent pathways may also take place [14]. Recently, the ring-like structure of D. radiodurans chromosomes was proposed to be a key to radioresistance in this bacterium [15]. Little has been known about the involvement of DNA damage response pathways accounting for the radioresistance in this organism. Our work details a novel mechanism by which D. radiodurans initiates its DNA damage response and provides evidence to support the role of specific downstream factors involved in DNA repair and damage avoidance. While studying DNA damage repair-deficient mutants of D. radiodurans, we isolated more than 40 natural mutant strains. KH8401 is one of the most sensitive mutant strains to DNA damaging agents. In this report, we describe the identification of a unique gene, pprI
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00965-3
Y. Hua et al. / Biochemical and Biophysical Research Communications 306 (2003) 354–360
(inducer of pleiotropic proteins promoting DNA repair), from this mutant. We also describe how the gene has been disrupted by a transposon and how this was used to verify the influence of the gene on DNA damage repair in the D. radiodurans mutant. It was confirmed that PprI, the gene product of pprI, serves as a general switch for downstream DNA repair and protection pathways via its regulatory function on the gene expression of recA, pprA (pleiotropic protein promoting DNA repair A.I. Narumi, unpublished work, see GenBank, Accession No. O32504), and enhancing the enzyme activities of catalases.
Methods Growth, radiation treatment, and survival curves of D. radiodurans. All D. radiodurans strains used in this work were grown at 30 °C in TGY medium or on TGY plates supplemented with 1.5% Bacto-agar. Two micrograms of streptomycin or 3 lg of chloramphenicol per ml was supplemented in the medium if necessary. D. radiodurans cells were grown to early stationary phase and used for determining the cell survival rate. For irradiation with c-rays, the cell suspension (1 ml) was irradiated at room temperature for 1 h with 60 Co c-rays at several different dosages (from 0.25 to 8 kGy), which were adjusted by changing the distance of samples from the c-ray source. After the treatments, the cells were diluted in phosphate buffer, plated on TGY plates, and incubated at 30 °C for 3 days prior to the colonies being enumerated. Cloning, disruption, and complementation of pprI. The unique NgoMI fragment existed in the mutant strain genome and was isolated and cloned into the pUC19 AvaI site. Candidate clones were collected after colony hybridization with probe IS8301 labeled with digoxigenin and further examined by subsequent PCR analysis. Clones containing the IS8301 sequence were used for DNA sequencing. The DNA sequence analysis was carried out using pUC19 universal primers and primers were designed based on the IS8301 sequence. Disruption of pprI was performed using the direct insertional mutagenesis technique described by Funayama et al. [16] with modifications. Briefly, a 8698-bp DNA fragment containing pprI was obtained from the cosmid pDC110 (the laboratory stock) by restricted digestion of ScaI. It was then ligated into the plasmid pUC19 pre-treated with HincII. Since pprI has a single AscI site the plasmid was digested with AscI; the linearized plasmid was then ligated to a 0.9-kb HincII fragment containing the chloramphenicol-resistance gene (catA) from pKatCAT [16]. This plasmid was linearized by PstI and transformed into D. radiodurans cells. Colonies able to grow on the TGY plate with chloramphenicol (3 lg/ml) were collected and further examined with PCR. Strains with the pprI (DR0167) disruption by the insertional mutagenesis were designated as YA. Complementation plasmids were constructed from pRADZ3 (a gift from Dr. M. Cox), a plasmid with GroES promoter, and markers, AmpRþ and ChmRþ . pRADZ3 was pre-treated with SpeI and NdeI. The PCR product of pprI (containing SpeI and NdeI builtin by its primers) was ligated to pGEM T-Easy vector (Promega, WI) and digested. The target gene pprI was then ligated to SpeI and NdeI-pre-digested pRADZ3, which resulted in pRADZ3pprI. To construct a kanamycin resistance plasmid, both pET-29b(+) (containing KanRþ ) and pRADZ3pprI were cut with AccI, their larger fragments were reclaimed and ligated, forming the pprI complementation plasmid, named pRADZKpprI, with a kanamycin resistance gene. The recA and pprA complementation plasmids, named pRADKrecA and pRADKpprA, were similarly constructed. The complementation plasmids were transformed into YA1. The trans-
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formants were obtained by kanamycin resistance selection and verified by sequencing. Biochemical characterization of RecA, PprA, and catalase. D. radiodurans cells were grown at 30 °C to early stationary phase and used for RecA and PprA Western blotting analysis. Cell suspension (1 ml) was irradiated at room temperature for 1 h with 60 Co c-rays at a dosage of 2 kGy. After a 1-h post-incubation, the irradiated cells were suspended in a phosphate buffer and disrupted with an ultrasonicator at 400 W output for a total of 6 min. The crude extracts were centrifuged (12,000g, 4 °C, 20 min) and the quantities of RecA and PprA proteins in the supernatants were measured by standard Western blotting analysis. To identify the downstream pathways that PprI regulates, two-dimensional (2D) gel electrophoresis was performed as described by G€ org et al. [17]. The radiation treatment conditions and the D. radiodurans crude extract preparation procedure are the same as described above. After 2D gel electrophoresis, the protein patterns of the wild-type and pprI-disruptant, with or without radiation treatment, were analyzed with the software supplied by the manufacturer (Amersham Biosciences, Uppsala state). Protein spots showing significant difference between wild-type and pprI-disruptant were excised from the gel and submitted for sequence identification by Edman sequencing or mass spectrometry. To detect the catalase activities, bacterial cellular extracts from YA1, KH8401A, KH8401M, and KD8301 were prepared and separated on non-denaturing polyacrylamide gels. Gels were stained for catalase activities by the horseradish peroxidase–diaminobenzidine method [18,19]. Measurement of the effects of D. radiodurans cell extracts on free radical induced DNA damage. D. radiodurans KD8301 and mutant YA1 cells were harvested at early stationary phase and disrupted by ultrasonication. The cell homogenates were centrifuged (12,000g, 4 °C, 20 min) to prepare the crude cell extracts. The inhibitory effect of cellular extracts on DNA damage was assayed using the Fenton reaction system with modification [20]. Intensity of luminescence was directly proportional to the amount of DNA damaged by free radicals. Briefly, the reaction was conducted by mixing the following reagents in the order: 100 ll of 2 104 mol/L CuSO4 , 70 ll of 2 103 mol/L ascorbic acid, 70 ll of 2 103 mol/L Phen, 440 ll of 0.1 mol/L acetate buffer (pH 5.5), 20 ll of 50 lg/ml calf thymus DNA (Sigma Chemical, USA), and 100 ll of cell extracts or distilled water for control experiment, and finally 200 ll of 0.8 mol/L H2 O2 was added to start the reaction. Intensity of luminescence counted over a 10 s period was monitored in an Ultra Weak Chemiluminescence Analyzer (Institute of Biophysics of Chinese Academy of Sciences, Beijing) at 37 °C. The measurements were triplicated and averaged. An inhibition ratio (I) was calculated by the following equation: I ð%Þ ¼ ½ðCLcontrol CL0 Þ ðCLsample CL0 Þ=ðCLcontrol CL0 Þ 100; where CLcontrol is the luminosity of control, CL0 is the luminosity of background, and CLsample is the luminosity of test samples.
Results We have isolated more than 40 different DNA damage repair-deficient mutant strains from D. radiodurans. Mutant strain KH8401 is one of the most radiosensitive strains, whose radioresistance is reduced by 2–3 orders of magnitude compared to wild-type D. radiodurans (Fig. 1A). Interestingly, after ionizing radiation the survival curve of this strain results in a long tail around the rate of 0.1%, which is suggestive of transposition. Even after repeated single colony selections, a small
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restriction endonucleases. Southern blotting showed that a 4.2-kb NgoMI fragment hybridized with transposon IS8301 and exists uniquely in all the radiosensitive colonies A, B, C, and D (Fig. 2A). The fragment was isolated and cloned into the pUC19 AvaI site. PCR and sequence analysis confirmed that the transposon disrupted the ORF01471 (DR0167) sequence in chromosome I of D. radiodurans, which encodes a novel ‘‘hypothetical protein,’’ responsible for loss of radioresistance. We designated DR0167 as inducer of pleiotropic protein for DNA repair (pprI). To confirm the insertion of IS8301 into the pprI locus in the genome of the KH8401 mutant strains, a genomic PCR was performed using a pair of primers derived from the ORF01471 sequence flanking the IS8301 (50 -A CGCCTAACAGAGGAATGCAGA-30 and 50 -GTTGC ACAGCGTTTCGATGACC-30 ). The primers were
Fig. 1. Radiosensitivity and reversion of the mutant D. radiodurans strain, KH8401. (A) Representative survival curves for D. radiodurans mutant strain KH8401 (open circles) and wild-type strain KD8301 (closed circles) following exposure to c-radiation. Values are means SD obtained from four different experiments. (B) DNA damage sensitivity of KH8401 and its derivatives. Data were obtained from triplicate experiments. Viable colonies after c- or UV-irradiation were designated as radioresistant colonies or revertants (e.g., M, N, O, and R).
number of cells could form colonies after ionizing irradiation or UV-treatment (Fig. 1B). The surviving colonies (e.g., M, N, O, and R of KH8401) were therefore designated radioresistant strains (revertants) and the dead colonies (e.g., A, B, C, and D of KH8401) radiosensitive strains (mutants). These results suggest that the radiation sensitive phenotype of strain KH8401 might be related to a transposition within a key radioresistance gene. To identify the insertion site, we have performed a genome wide search. All known transposon sequences of D. radiodurans were used as probes to detect the insertion site within genomic DNA digested by different
Fig. 2. Localization of the transposon IS8301 in the genome of radiosensitive mutant, KH8401. (A) The distribution profile of transposons in the genome of D. radiodurans by a genomic Southern blot. The genomic DNA was isolated from both mutant strains (KH8401A, B, C, and D) and revertant strains (KH8401M, N, O, and R) and digested with NgoMI. Samples were electrophoresed and hybridized with digoxigenin-labeled IS8301. The arrow indicates the extra band present in the genome of the mutant colonies. (B) The insertion of IS8301 into the genome of KH8401 mutant colonies examined by a genomic PCR using a pair of primers derived from pprI (DR0167). PCR products were observed at a length of 600-bp in the revertants (KH8401M, N, O, and R) and all wild strains (1, ATCC/R1; 2, KD8301; 3, KN101; 4, KR1-1; 5, KR1-2; and 6, MR1), but a 2.4-kb band was observed in all mutant colonies (KH8401A, B, C, and D) analyzed.
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designed to give a PCR product 596 bp in length in wildtype strains and a longer fragment in mutants. The PCR results revealed a 2.4-kb band in all 4 mutant colonies (KH8401 A, B, C, and D), whereas a 600-bp band was observed in the revertant colonies (KH8401 M, N, O, and R) and all wild type strains (KD8301, KN101, KR1-1, KR1-2, and MR1) (Fig. 2B). The size difference between the two groups of PCR products correlates well with the calculated size of the IS8301 (1.8 kb), confirming that pprI in the mutants was disrupted by transposon IS8301 and that the transposon was eliminated in each of the revertant strains. To further examine the physiological function of the gene, pprI disruptants were constructed by transforming a linearized disruption plasmid, pprI536::cat (Fig. 3A), into KD8301 (YA1, YA2, and YA3). By means of genomic PCR, we confirmed that the cat gene (chloramphenicol-resistance gene) of 915 bp length was successfully inserted into pprI of KD8301 (Fig. 3B). Following 1-h c-ray irradiation a survival curve of the disruptant YA1 was taken. The disruptants, such as YA1, were much more sensitive to c-ray irradiation than the natural mutant KH8401 and did not display a long ‘‘tail,’’ indicating that the disruption causes a complete elimination of the PprI function (Fig. 3C). Since either transpositional mutation or disruption can eliminate the function of PprI, the radioresistance phenotype should be restored by functional complementation with expression of the pprI gene on a plasmid. We expressed pprI in the pprI disruptant using the complementation plasmid, pRADZKpprI, based on plasmid pRADZ3. We found that the radioresistance of the disruptant could be fully restored by complementation with pprI (Fig. 3C). To identify the downstream pathways that PprI regulates, we performed a genome wide search for the proteins whose expression profiles under radiation stress are controlled by PprI. About 10 peptides were observed in 2D gel electrophoresis. These peptides were overexpressed after irradiation, but their differential expression disappeared in mutant strains lacking functional PprI even under the stress exposure. RecA and PprA are two of such proteins identified. To verify the 2D gel electrophoresis results, Western blotting analysis was employed using antibodies against RecA and PprA and crude extracts were prepared from wild-type (KD8301), mutant (KH8401A), revertant (KH8401M), and disruptant (YA1), with or without c-ray irradiation. Following ionizing irradiation, induced expression of both recA and pprA was observed in both KD8301 and KH8401M, whereas there were no changes in the intracellular levels of RecA and PprA in KH8401A and YA1 (Fig. 4A). In addition, when the pprI disruptant was complemented by recA or pprA, the radioresistance of the disruptant could only be partially restored (Fig. 3C). This suggests that additional factors play a role in the PprI pathway-mediated radioresistance.
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Fig. 3. Disruption and characterization of the D. radiodurans pprI gene functions. (A) Strategy of the pprI gene disruption. The two large arrows indicate the position and orientation of pprI and catA genes, respectively. The small arrows indicate the positions of specific primers used for PCR. (B) Colonies from the chloramphenicol selection were examined by genomic PCR. The PCR products of KD8301 revealed a band length of 500 bp (lane 4), whereas those of YA1, YA2, and YA3 (lanes 1–3) resulted in a 1.5-kb band. M denotes molecular markers. (C) Representative survival curves for D. radiodurans disruptant YA1 (closed triangles), mutant KH8401 (closed squares), and wild-type KD8301 (closed circles), and changes in survival rates due to the complementation by PprI (open squares), RecA (open triangles), and PprA (open circles) in the disruptant, following exposure to c-radiation. Values are means SD obtained from four independent experiments.
We provide further evidence indicating that PprI regulates the enzyme activities of free radical ‘‘scavenger proteins.’’ The effects of KD8301 and YA1 cellular extracts on free radical induced DNA damage were first examined by using Fenton reaction system [15]. Wild type PprI remarkably suppressed chemiluminescence of
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PprI has a regulatory function on the enzyme activities of the free radical ‘‘scavenger proteins.’’
Discussion
Fig. 4. Regulation of recA and pprA expression and catalase activities by PprI. (A) KD8301, KH8401A, KH8401M, and YA1 cell extracts were treated (+) or mock treated ()) with 60 Co c-radiation at a dose rate of 2 kGy for 1 h and analyzed by Western blot using anti-RecA and anti-PprA. Anti-GroEL was used as the sample loading control. The bottom panel shows the semi-quantitation of Western blotting of RecA and PprA before and after irradiation. The intensity of the band in KD8301 before irradiation was arbitrarily taken as 1. (B) Defectiveness of PprI in KD8301 (closed circles), YA1 (closed triangles), and mock control (open circles) on the DNA protection from free radicals. (C) Catalase A and B activities in cell extracts of KD8301 (lane 1) and KH8401M (lane 3), compared to those of KH8401A (lane 2) and YA1 (lane 4) D. radiodurans strains. Anti-GroEL Western blotting bands were used for sample loading control.
free radical-induced DNA damage with an inhibition ratio of 93.10%, while the disruption of the gene reduced the inhibition ratio to 59.91% (Fig. 4B). Catalase was considered as one of the major enzymes in D. radiodurans, preventing DNA damage from hydrogen peroxide and oxygen free radicals generated by ionizing radiation [14–16]. Comparing with those of the wild-type KD8301 and pprI-revertant KH8401M, the enzymatic activities of the pprI-disruptant YA1 and mutant KH8401A were analyzed. The defect in pprI caused significantly reduced catalase activities in the disruptant and mutant strains (2–3-fold decrease, Fig. 4C). These results suggest that
It has been observed that D. radiodurans, a nonspore-forming bacterium, has the highest tolerance to ionizing radiation-caused DNA double-strand breaks [21]. D. radiodurans is a multigenomic organism, having as many as 10 genome copies per cell [22]. It was predicted that D. radiodurans uses this genetic redundancy and homologous recombination to reassemble its genome following irradiation [8]. Unfortunately, detailed examinations of the genomic DNA sequence of D. radiodurans R1 have failed to provide any insights that help to explain the DNA damage tolerance of this species [5,6]. However, it is shown that, of the 3187 open reading frames identified in the D. radiodurans genome, only 1493 could be assigned a function based on similarity to other gene products found in the protein database [5]. To date, of the remaining 1694 proteins of unknown function, about 1000 are unique to D. radiodurans, showing no database match and implying that D. radiodurans has unprecedented mechanisms for facilitating the cellÕs recovery from DNA damage. The hypothetical proteins encoded by the D. radiodurans genome are therefore a good source for discovering evolved, highly specific, and distinctive mechanisms to deal with DNA damage. From D. radiodurans KH8401, a natural mutant showing a unique phenotype following irradiation, we were able to identify such a protein, PprI. It is a general switch responsible for extreme radioresistance. PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. Recently, it was shown that the D. radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. It has been proposed that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroid, radiation-generated free DNA ends are held together, which may facilitate template-independent yet errorfree joining of DNA breaks [15]. However, a recA-defective D. radiodurans mutant, which is highly sensitive to both UV and ionizing radiation [14], also exhibited a toroidal DNA morphology. In contrast to wild-type cells, the recA mutant showed no evidence of DNA spreading between compartment or nucleoid fusion [15], indicating that both RecA and the toroidal DNA morphology contribute to the radioresistance in D. radiodurans. We believe that there are a number of ways that proteins might help reconstruct an intact genome from fragments that remain after c-irradiation. The chromosomal ring-like-structure and the PprImediated DNA repair and protection are two
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independent and important pathways for maintaining genomic integrity in D. radiodurans. A homology search across all genomic databases revealed no open reading frame with significant sequence similarity to PprI, suggesting it is a unique gene in D. radiodurans. However, PprI protein shows sequence similarity to two functional motifs: neutral zinc metallopeptidase (zinc-binding signature) and bacterial regulatory protein (lacI family signature), indicating that this protein has regulatory functions [7]. This gene has also been recently identified and named irrE by the Battista laboratory using a different strategy [23]. Complete deletion of the PprI function eliminates the induction of recA gene expression. RecA is required only for the repair of extreme DNA damage in D. radiodurans and it is expressed only when DNA is severely damaged [24]. Significant induction of recA expression following DNA damage has been observed [25,26]. PprA is a major DNA repair gene involved in a RecA-independent pathway (unpublished data) and is another gene whose expression is strictly regulated by PprI. Protective mechanisms against oxidative damage may also contribute to its extreme radiation resistance, since the lethal effect of ionizing radiation is known to be caused by oxygen-enhanced generation of hydrogen peroxide and oxygen free radicals, which damage cell membranes, proteins, and nucleic acids [27]. It has been shown that a pre-treatment with H2 O2 of D. radiodurans cultures at early exponential-growth phase enhances radiation resistance [28]. Moreover, D. radiodurans expresses relatively high levels of catalase activities [29]. Both null mutants have been shown to be significantly more sensitive to ionizing radiation [30]. We show here that the antioxidant activities of catalases were significantly reduced in the pprI-disruptant and mutant strains. Response to DNA damage is mediated by a signal transduction pathway consisting of sensors, transducers, and effectors [31]. DNA damage is detected by sensor proteins, which in turn activate PprI, a major signal transducer. Activated PprI, as a general switch, regulates the expression and activation of a number of DNA repair and protection genes, including recA, pprA, and catalases. Although PprI is unique to D. radiodurans, finding its functional homologues and characterization of their shared mechanism in eukaryotic systems will contribute to the deeper understanding of radiation damage, repair, and resistance, as well as to the improvement of human health. Acknowledgments This work was supported by a grant of the National Natural Science Foundation of China to Y.J.H., and as part of a Cross Over Project of the Ministry of Industrial Science and Technology, Japan, and partially supported by a NIH grant to B.H.S. We thank Dr. Mike Cox, Department of Biochemistry, University of Wisconsin-Madison,
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for his generous gift of the plasmid pRADZ3, and Dr. Steve Alas for critical reading of the manuscript.
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PprI: a general switch responsible for extreme radioresistance of Deinococcus radioduransq Yuejin Hua,a,b,* Issay Narumi,b Guanjun Gao,a Bing Tian,a Katsuya Satoh,b Shigeru Kitayama,b and Binghui Shenc b
a Institute of Nuclear-Agricultural Science, Zhejiang University, Hangzhou 310029, China Biotechnology Laboratory, Takasaki Radiochemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki 370-1292, Japan c Division of Molecular Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010-3000, USA
Received 5 May 2003
Abstract Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress. Ó 2003 Elsevier Science (USA). All rights reserved.
The extremely radioresistant bacterium, Deinococcus radiodurans, is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet radiation, and desiccation [1,2]. Its unprecedented ability to avoid DNA damage in the presence of heavy irradiation has made it a critical model for pursuing genes involved in maintaining DNA integrity and stability. For decades, scientists have been attempting to delineate the mechanisms by which this bacterium overcomes these extreme levels of DNA damage-inducing agents. The bacteriumÕs phenomenal radioresistance was found to derive from its ability to repair hundreds of double-strand DNA breaks [3,4]. D. radiodurans is able to reconstruct a functional genome from chromosomal fragments, q
Abbreviations: PprI, inducer of pleiotropic proteins promoting DNA repair; PprA, pleiotropic protein promoting DNA repair A; RecA, recombinase A. * Corresponding author. Fax: +86-571-8697-1703. E-mail address: [email protected] (Y. Hua).
whereas the genomes of most organisms are irreversibly shattered by the effects of high levels of ionizing radiation [5–9]. This process was thought to occur mainly via homologous recombination requiring RecA (recombinase A) function [10–13], although DNA repair via other RecA-independent pathways may also take place [14]. Recently, the ring-like structure of D. radiodurans chromosomes was proposed to be a key to radioresistance in this bacterium [15]. Little has been known about the involvement of DNA damage response pathways accounting for the radioresistance in this organism. Our work details a novel mechanism by which D. radiodurans initiates its DNA damage response and provides evidence to support the role of specific downstream factors involved in DNA repair and damage avoidance. While studying DNA damage repair-deficient mutants of D. radiodurans, we isolated more than 40 natural mutant strains. KH8401 is one of the most sensitive mutant strains to DNA damaging agents. In this report, we describe the identification of a unique gene, pprI
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00965-3
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(inducer of pleiotropic proteins promoting DNA repair), from this mutant. We also describe how the gene has been disrupted by a transposon and how this was used to verify the influence of the gene on DNA damage repair in the D. radiodurans mutant. It was confirmed that PprI, the gene product of pprI, serves as a general switch for downstream DNA repair and protection pathways via its regulatory function on the gene expression of recA, pprA (pleiotropic protein promoting DNA repair A.I. Narumi, unpublished work, see GenBank, Accession No. O32504), and enhancing the enzyme activities of catalases.
Methods Growth, radiation treatment, and survival curves of D. radiodurans. All D. radiodurans strains used in this work were grown at 30 °C in TGY medium or on TGY plates supplemented with 1.5% Bacto-agar. Two micrograms of streptomycin or 3 lg of chloramphenicol per ml was supplemented in the medium if necessary. D. radiodurans cells were grown to early stationary phase and used for determining the cell survival rate. For irradiation with c-rays, the cell suspension (1 ml) was irradiated at room temperature for 1 h with 60 Co c-rays at several different dosages (from 0.25 to 8 kGy), which were adjusted by changing the distance of samples from the c-ray source. After the treatments, the cells were diluted in phosphate buffer, plated on TGY plates, and incubated at 30 °C for 3 days prior to the colonies being enumerated. Cloning, disruption, and complementation of pprI. The unique NgoMI fragment existed in the mutant strain genome and was isolated and cloned into the pUC19 AvaI site. Candidate clones were collected after colony hybridization with probe IS8301 labeled with digoxigenin and further examined by subsequent PCR analysis. Clones containing the IS8301 sequence were used for DNA sequencing. The DNA sequence analysis was carried out using pUC19 universal primers and primers were designed based on the IS8301 sequence. Disruption of pprI was performed using the direct insertional mutagenesis technique described by Funayama et al. [16] with modifications. Briefly, a 8698-bp DNA fragment containing pprI was obtained from the cosmid pDC110 (the laboratory stock) by restricted digestion of ScaI. It was then ligated into the plasmid pUC19 pre-treated with HincII. Since pprI has a single AscI site the plasmid was digested with AscI; the linearized plasmid was then ligated to a 0.9-kb HincII fragment containing the chloramphenicol-resistance gene (catA) from pKatCAT [16]. This plasmid was linearized by PstI and transformed into D. radiodurans cells. Colonies able to grow on the TGY plate with chloramphenicol (3 lg/ml) were collected and further examined with PCR. Strains with the pprI (DR0167) disruption by the insertional mutagenesis were designated as YA. Complementation plasmids were constructed from pRADZ3 (a gift from Dr. M. Cox), a plasmid with GroES promoter, and markers, AmpRþ and ChmRþ . pRADZ3 was pre-treated with SpeI and NdeI. The PCR product of pprI (containing SpeI and NdeI builtin by its primers) was ligated to pGEM T-Easy vector (Promega, WI) and digested. The target gene pprI was then ligated to SpeI and NdeI-pre-digested pRADZ3, which resulted in pRADZ3pprI. To construct a kanamycin resistance plasmid, both pET-29b(+) (containing KanRþ ) and pRADZ3pprI were cut with AccI, their larger fragments were reclaimed and ligated, forming the pprI complementation plasmid, named pRADZKpprI, with a kanamycin resistance gene. The recA and pprA complementation plasmids, named pRADKrecA and pRADKpprA, were similarly constructed. The complementation plasmids were transformed into YA1. The trans-
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formants were obtained by kanamycin resistance selection and verified by sequencing. Biochemical characterization of RecA, PprA, and catalase. D. radiodurans cells were grown at 30 °C to early stationary phase and used for RecA and PprA Western blotting analysis. Cell suspension (1 ml) was irradiated at room temperature for 1 h with 60 Co c-rays at a dosage of 2 kGy. After a 1-h post-incubation, the irradiated cells were suspended in a phosphate buffer and disrupted with an ultrasonicator at 400 W output for a total of 6 min. The crude extracts were centrifuged (12,000g, 4 °C, 20 min) and the quantities of RecA and PprA proteins in the supernatants were measured by standard Western blotting analysis. To identify the downstream pathways that PprI regulates, two-dimensional (2D) gel electrophoresis was performed as described by G€ org et al. [17]. The radiation treatment conditions and the D. radiodurans crude extract preparation procedure are the same as described above. After 2D gel electrophoresis, the protein patterns of the wild-type and pprI-disruptant, with or without radiation treatment, were analyzed with the software supplied by the manufacturer (Amersham Biosciences, Uppsala state). Protein spots showing significant difference between wild-type and pprI-disruptant were excised from the gel and submitted for sequence identification by Edman sequencing or mass spectrometry. To detect the catalase activities, bacterial cellular extracts from YA1, KH8401A, KH8401M, and KD8301 were prepared and separated on non-denaturing polyacrylamide gels. Gels were stained for catalase activities by the horseradish peroxidase–diaminobenzidine method [18,19]. Measurement of the effects of D. radiodurans cell extracts on free radical induced DNA damage. D. radiodurans KD8301 and mutant YA1 cells were harvested at early stationary phase and disrupted by ultrasonication. The cell homogenates were centrifuged (12,000g, 4 °C, 20 min) to prepare the crude cell extracts. The inhibitory effect of cellular extracts on DNA damage was assayed using the Fenton reaction system with modification [20]. Intensity of luminescence was directly proportional to the amount of DNA damaged by free radicals. Briefly, the reaction was conducted by mixing the following reagents in the order: 100 ll of 2 104 mol/L CuSO4 , 70 ll of 2 103 mol/L ascorbic acid, 70 ll of 2 103 mol/L Phen, 440 ll of 0.1 mol/L acetate buffer (pH 5.5), 20 ll of 50 lg/ml calf thymus DNA (Sigma Chemical, USA), and 100 ll of cell extracts or distilled water for control experiment, and finally 200 ll of 0.8 mol/L H2 O2 was added to start the reaction. Intensity of luminescence counted over a 10 s period was monitored in an Ultra Weak Chemiluminescence Analyzer (Institute of Biophysics of Chinese Academy of Sciences, Beijing) at 37 °C. The measurements were triplicated and averaged. An inhibition ratio (I) was calculated by the following equation: I ð%Þ ¼ ½ðCLcontrol CL0 Þ ðCLsample CL0 Þ=ðCLcontrol CL0 Þ 100; where CLcontrol is the luminosity of control, CL0 is the luminosity of background, and CLsample is the luminosity of test samples.
Results We have isolated more than 40 different DNA damage repair-deficient mutant strains from D. radiodurans. Mutant strain KH8401 is one of the most radiosensitive strains, whose radioresistance is reduced by 2–3 orders of magnitude compared to wild-type D. radiodurans (Fig. 1A). Interestingly, after ionizing radiation the survival curve of this strain results in a long tail around the rate of 0.1%, which is suggestive of transposition. Even after repeated single colony selections, a small
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restriction endonucleases. Southern blotting showed that a 4.2-kb NgoMI fragment hybridized with transposon IS8301 and exists uniquely in all the radiosensitive colonies A, B, C, and D (Fig. 2A). The fragment was isolated and cloned into the pUC19 AvaI site. PCR and sequence analysis confirmed that the transposon disrupted the ORF01471 (DR0167) sequence in chromosome I of D. radiodurans, which encodes a novel ‘‘hypothetical protein,’’ responsible for loss of radioresistance. We designated DR0167 as inducer of pleiotropic protein for DNA repair (pprI). To confirm the insertion of IS8301 into the pprI locus in the genome of the KH8401 mutant strains, a genomic PCR was performed using a pair of primers derived from the ORF01471 sequence flanking the IS8301 (50 -A CGCCTAACAGAGGAATGCAGA-30 and 50 -GTTGC ACAGCGTTTCGATGACC-30 ). The primers were
Fig. 1. Radiosensitivity and reversion of the mutant D. radiodurans strain, KH8401. (A) Representative survival curves for D. radiodurans mutant strain KH8401 (open circles) and wild-type strain KD8301 (closed circles) following exposure to c-radiation. Values are means SD obtained from four different experiments. (B) DNA damage sensitivity of KH8401 and its derivatives. Data were obtained from triplicate experiments. Viable colonies after c- or UV-irradiation were designated as radioresistant colonies or revertants (e.g., M, N, O, and R).
number of cells could form colonies after ionizing irradiation or UV-treatment (Fig. 1B). The surviving colonies (e.g., M, N, O, and R of KH8401) were therefore designated radioresistant strains (revertants) and the dead colonies (e.g., A, B, C, and D of KH8401) radiosensitive strains (mutants). These results suggest that the radiation sensitive phenotype of strain KH8401 might be related to a transposition within a key radioresistance gene. To identify the insertion site, we have performed a genome wide search. All known transposon sequences of D. radiodurans were used as probes to detect the insertion site within genomic DNA digested by different
Fig. 2. Localization of the transposon IS8301 in the genome of radiosensitive mutant, KH8401. (A) The distribution profile of transposons in the genome of D. radiodurans by a genomic Southern blot. The genomic DNA was isolated from both mutant strains (KH8401A, B, C, and D) and revertant strains (KH8401M, N, O, and R) and digested with NgoMI. Samples were electrophoresed and hybridized with digoxigenin-labeled IS8301. The arrow indicates the extra band present in the genome of the mutant colonies. (B) The insertion of IS8301 into the genome of KH8401 mutant colonies examined by a genomic PCR using a pair of primers derived from pprI (DR0167). PCR products were observed at a length of 600-bp in the revertants (KH8401M, N, O, and R) and all wild strains (1, ATCC/R1; 2, KD8301; 3, KN101; 4, KR1-1; 5, KR1-2; and 6, MR1), but a 2.4-kb band was observed in all mutant colonies (KH8401A, B, C, and D) analyzed.
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designed to give a PCR product 596 bp in length in wildtype strains and a longer fragment in mutants. The PCR results revealed a 2.4-kb band in all 4 mutant colonies (KH8401 A, B, C, and D), whereas a 600-bp band was observed in the revertant colonies (KH8401 M, N, O, and R) and all wild type strains (KD8301, KN101, KR1-1, KR1-2, and MR1) (Fig. 2B). The size difference between the two groups of PCR products correlates well with the calculated size of the IS8301 (1.8 kb), confirming that pprI in the mutants was disrupted by transposon IS8301 and that the transposon was eliminated in each of the revertant strains. To further examine the physiological function of the gene, pprI disruptants were constructed by transforming a linearized disruption plasmid, pprI536::cat (Fig. 3A), into KD8301 (YA1, YA2, and YA3). By means of genomic PCR, we confirmed that the cat gene (chloramphenicol-resistance gene) of 915 bp length was successfully inserted into pprI of KD8301 (Fig. 3B). Following 1-h c-ray irradiation a survival curve of the disruptant YA1 was taken. The disruptants, such as YA1, were much more sensitive to c-ray irradiation than the natural mutant KH8401 and did not display a long ‘‘tail,’’ indicating that the disruption causes a complete elimination of the PprI function (Fig. 3C). Since either transpositional mutation or disruption can eliminate the function of PprI, the radioresistance phenotype should be restored by functional complementation with expression of the pprI gene on a plasmid. We expressed pprI in the pprI disruptant using the complementation plasmid, pRADZKpprI, based on plasmid pRADZ3. We found that the radioresistance of the disruptant could be fully restored by complementation with pprI (Fig. 3C). To identify the downstream pathways that PprI regulates, we performed a genome wide search for the proteins whose expression profiles under radiation stress are controlled by PprI. About 10 peptides were observed in 2D gel electrophoresis. These peptides were overexpressed after irradiation, but their differential expression disappeared in mutant strains lacking functional PprI even under the stress exposure. RecA and PprA are two of such proteins identified. To verify the 2D gel electrophoresis results, Western blotting analysis was employed using antibodies against RecA and PprA and crude extracts were prepared from wild-type (KD8301), mutant (KH8401A), revertant (KH8401M), and disruptant (YA1), with or without c-ray irradiation. Following ionizing irradiation, induced expression of both recA and pprA was observed in both KD8301 and KH8401M, whereas there were no changes in the intracellular levels of RecA and PprA in KH8401A and YA1 (Fig. 4A). In addition, when the pprI disruptant was complemented by recA or pprA, the radioresistance of the disruptant could only be partially restored (Fig. 3C). This suggests that additional factors play a role in the PprI pathway-mediated radioresistance.
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Fig. 3. Disruption and characterization of the D. radiodurans pprI gene functions. (A) Strategy of the pprI gene disruption. The two large arrows indicate the position and orientation of pprI and catA genes, respectively. The small arrows indicate the positions of specific primers used for PCR. (B) Colonies from the chloramphenicol selection were examined by genomic PCR. The PCR products of KD8301 revealed a band length of 500 bp (lane 4), whereas those of YA1, YA2, and YA3 (lanes 1–3) resulted in a 1.5-kb band. M denotes molecular markers. (C) Representative survival curves for D. radiodurans disruptant YA1 (closed triangles), mutant KH8401 (closed squares), and wild-type KD8301 (closed circles), and changes in survival rates due to the complementation by PprI (open squares), RecA (open triangles), and PprA (open circles) in the disruptant, following exposure to c-radiation. Values are means SD obtained from four independent experiments.
We provide further evidence indicating that PprI regulates the enzyme activities of free radical ‘‘scavenger proteins.’’ The effects of KD8301 and YA1 cellular extracts on free radical induced DNA damage were first examined by using Fenton reaction system [15]. Wild type PprI remarkably suppressed chemiluminescence of
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PprI has a regulatory function on the enzyme activities of the free radical ‘‘scavenger proteins.’’
Discussion
Fig. 4. Regulation of recA and pprA expression and catalase activities by PprI. (A) KD8301, KH8401A, KH8401M, and YA1 cell extracts were treated (+) or mock treated ()) with 60 Co c-radiation at a dose rate of 2 kGy for 1 h and analyzed by Western blot using anti-RecA and anti-PprA. Anti-GroEL was used as the sample loading control. The bottom panel shows the semi-quantitation of Western blotting of RecA and PprA before and after irradiation. The intensity of the band in KD8301 before irradiation was arbitrarily taken as 1. (B) Defectiveness of PprI in KD8301 (closed circles), YA1 (closed triangles), and mock control (open circles) on the DNA protection from free radicals. (C) Catalase A and B activities in cell extracts of KD8301 (lane 1) and KH8401M (lane 3), compared to those of KH8401A (lane 2) and YA1 (lane 4) D. radiodurans strains. Anti-GroEL Western blotting bands were used for sample loading control.
free radical-induced DNA damage with an inhibition ratio of 93.10%, while the disruption of the gene reduced the inhibition ratio to 59.91% (Fig. 4B). Catalase was considered as one of the major enzymes in D. radiodurans, preventing DNA damage from hydrogen peroxide and oxygen free radicals generated by ionizing radiation [14–16]. Comparing with those of the wild-type KD8301 and pprI-revertant KH8401M, the enzymatic activities of the pprI-disruptant YA1 and mutant KH8401A were analyzed. The defect in pprI caused significantly reduced catalase activities in the disruptant and mutant strains (2–3-fold decrease, Fig. 4C). These results suggest that
It has been observed that D. radiodurans, a nonspore-forming bacterium, has the highest tolerance to ionizing radiation-caused DNA double-strand breaks [21]. D. radiodurans is a multigenomic organism, having as many as 10 genome copies per cell [22]. It was predicted that D. radiodurans uses this genetic redundancy and homologous recombination to reassemble its genome following irradiation [8]. Unfortunately, detailed examinations of the genomic DNA sequence of D. radiodurans R1 have failed to provide any insights that help to explain the DNA damage tolerance of this species [5,6]. However, it is shown that, of the 3187 open reading frames identified in the D. radiodurans genome, only 1493 could be assigned a function based on similarity to other gene products found in the protein database [5]. To date, of the remaining 1694 proteins of unknown function, about 1000 are unique to D. radiodurans, showing no database match and implying that D. radiodurans has unprecedented mechanisms for facilitating the cellÕs recovery from DNA damage. The hypothetical proteins encoded by the D. radiodurans genome are therefore a good source for discovering evolved, highly specific, and distinctive mechanisms to deal with DNA damage. From D. radiodurans KH8401, a natural mutant showing a unique phenotype following irradiation, we were able to identify such a protein, PprI. It is a general switch responsible for extreme radioresistance. PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. Recently, it was shown that the D. radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. It has been proposed that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroid, radiation-generated free DNA ends are held together, which may facilitate template-independent yet errorfree joining of DNA breaks [15]. However, a recA-defective D. radiodurans mutant, which is highly sensitive to both UV and ionizing radiation [14], also exhibited a toroidal DNA morphology. In contrast to wild-type cells, the recA mutant showed no evidence of DNA spreading between compartment or nucleoid fusion [15], indicating that both RecA and the toroidal DNA morphology contribute to the radioresistance in D. radiodurans. We believe that there are a number of ways that proteins might help reconstruct an intact genome from fragments that remain after c-irradiation. The chromosomal ring-like-structure and the PprImediated DNA repair and protection are two
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independent and important pathways for maintaining genomic integrity in D. radiodurans. A homology search across all genomic databases revealed no open reading frame with significant sequence similarity to PprI, suggesting it is a unique gene in D. radiodurans. However, PprI protein shows sequence similarity to two functional motifs: neutral zinc metallopeptidase (zinc-binding signature) and bacterial regulatory protein (lacI family signature), indicating that this protein has regulatory functions [7]. This gene has also been recently identified and named irrE by the Battista laboratory using a different strategy [23]. Complete deletion of the PprI function eliminates the induction of recA gene expression. RecA is required only for the repair of extreme DNA damage in D. radiodurans and it is expressed only when DNA is severely damaged [24]. Significant induction of recA expression following DNA damage has been observed [25,26]. PprA is a major DNA repair gene involved in a RecA-independent pathway (unpublished data) and is another gene whose expression is strictly regulated by PprI. Protective mechanisms against oxidative damage may also contribute to its extreme radiation resistance, since the lethal effect of ionizing radiation is known to be caused by oxygen-enhanced generation of hydrogen peroxide and oxygen free radicals, which damage cell membranes, proteins, and nucleic acids [27]. It has been shown that a pre-treatment with H2 O2 of D. radiodurans cultures at early exponential-growth phase enhances radiation resistance [28]. Moreover, D. radiodurans expresses relatively high levels of catalase activities [29]. Both null mutants have been shown to be significantly more sensitive to ionizing radiation [30]. We show here that the antioxidant activities of catalases were significantly reduced in the pprI-disruptant and mutant strains. Response to DNA damage is mediated by a signal transduction pathway consisting of sensors, transducers, and effectors [31]. DNA damage is detected by sensor proteins, which in turn activate PprI, a major signal transducer. Activated PprI, as a general switch, regulates the expression and activation of a number of DNA repair and protection genes, including recA, pprA, and catalases. Although PprI is unique to D. radiodurans, finding its functional homologues and characterization of their shared mechanism in eukaryotic systems will contribute to the deeper understanding of radiation damage, repair, and resistance, as well as to the improvement of human health. Acknowledgments This work was supported by a grant of the National Natural Science Foundation of China to Y.J.H., and as part of a Cross Over Project of the Ministry of Industrial Science and Technology, Japan, and partially supported by a NIH grant to B.H.S. We thank Dr. Mike Cox, Department of Biochemistry, University of Wisconsin-Madison,
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for his generous gift of the plasmid pRADZ3, and Dr. Steve Alas for critical reading of the manuscript.
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