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1 Gene Resource Research Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki 370-1292, Japan
2 DNA Repair Protein Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki 370-1292, Japan
3 Material Science Laboratory, Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu 376-8515, Japan
Correspondence
Katsuya Satoh
sato.katsuya{at}jaea.go.jp
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ABSTRACT |
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-irradiation as in the wild-type strain. These results indicated that the two LexA homologues did not possess functional overlap regarding the induction of RecA. The lexA2 disruptant strains exhibited a much higher resistance to -rays than the wild-type strain. Furthermore, a luciferase assay showed that pprA promoter activation was enhanced in the lexA2 disruptant strain following -irradiation. The pprA gene encoding the novel radiation-inducible protein PprA plays a critical role in the radioresistance of D. radiodurans. The increase in radioresistance of the lexA2 disruptant strain is explained in part by the enhancement of pprA promoter activation.
The GenBank/EMBL/DDBJ accession number for the lexA2 sequence reported in this paper is AB256032.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Narumi, 2003; Cox & Battista, 2005). The most noteworthy characteristic of D. radiodurans is its capacity for repairing DNA double-strand breaks induced by ionizing radiation (Dean et al., 1966; Kitayama & Matsuyama, 1968; Grimsley et al., 1991). The highly efficient double-strand break repair process in D. radiodurans is radiation-inducible and is dependent on de novo protein synthesis following irradiation (Kitayama & Matsuyama, 1971). Several DNA-damage-inducible proteins that may be necessary for DNA repair have been detected in cell extracts of D. radiodurans using 2D-PAGE analysis (Hansen, 1980; Tanaka et al., 1996). Recent transcriptome and proteome analyses have revealed the comprehensive expression profiles of radiation-inducible genes and proteins (Lipton et al., 2002; Liu et al., 2003; Tanaka et al., 2004; Zhang et al., 2005). It has therefore been established that D. radiodurans possesses a DNA damage response mechanism. Additionally, analysis of DNA-damage-sensitive strains IRS24 (Mattimore et al., 1995) and KH840 (Kitayama & Matsuyama, 1975) identified a novel regulatory protein (DR0167; PprI protein, also called IrrE protein) that plays a role in regulating recA and pprA gene expression following -irradiation (Earl et al., 2002; Hua et al., 2003; Ohba et al., 2005). Furthermore, in a previous study we identified the radiation-responsive minimal promoter of the pprA gene encoding the novel radiation-inducible protein PprA, which plays a critical role in radioresistance of D. radiodurans (Narumi et al., 2004; Ohba et al., 2005).
RecA and LexA proteins in Escherichia coli play important roles in the DNA damage response repair mechanism (the SOS system) (Walker, 1984; Friedberg et al., 1995). The SOS response in Bacillus subtilis progresses in a similar manner, with B. subtilis RecA having an identical role in controlling the SOS regulon together with a cellular repressor protein that is functionally homologous to the E. coli LexA repressor (Wojciechowski et al., 1991). SOS-like processes have been conserved in a wide variety of eubacterial species (Miller & Kokjohn, 1990; Roca & Cox, 1990). On the one hand, RecA is the sole protein required for LexA1 (DRA0344) cleavage, although LexA1 is not involved in RecA induction in D. radiodurans (Narumi et al., 2001; Bonacossa de Almeida et al., 2002). The D. radiodurans DNA-repair-deficient mutant KI696 carrying the recA424 mutation was only slightly sensitive to -rays in comparison with the wild-type. RecA424 completely lacked the recombination activity but retained the co-protease activity. On the basis of this finding we suggested that the co-protease activity rather than the recombination activity of RecA contributed to the extraordinary radiation resistance of D. radiodurans (Satoh et al., 2002). Bioinformatic analysis of the D. radiodurans genome sequence showed that the genome encodes a second, diverged copy of LexA1, LexA2 (DRA0074), which retains the same arrangement of the helix–turn–helix DNA-binding domain and the autocleavage domain (Makarova et al., 2001). Sheng et al. (2004) suggested that the product of gene dra0074 is involved in metabolic pathways other than the DNA repair response elicited by irradiation. However, the exact role of LexA2 and the relationship between LexA1 and LexA2 in the radiation response are poorly understood. The biochemical function of LexA2 therefore requires further clarification.
This study began with the purpose of elucidating the role of LexA2 in relation to the radiation response mechanism of D. radiodurans. To accomplish this purpose, we generated lexA1 and lexA2 disruptant strains in wild-type and recA backgrounds. Our results suggest that LexA2 contributes to the regulation of the radiation response promoter of the pprA gene in D. radiodurans and that it is not involved in RecA induction.
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METHODS |
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. D. radiodurans cells were grown with agitation at 30 °C in TGY broth, containing 0.5 % (w/v) tryptone-peptone, 0.1 % (w/v) glucose and 0.3 % (w/v) yeast extract, or on TGY agar solidified with 1.5 % (w/v) agar. When necessary, 3 µg chloramphenicol ml–1, 16 µg kanamycin ml–1 and 350 µg spectinomycin ml–1 were added. E. coli strains were grown in Luria–Bertani (LB) broth-Miller or on LB agar-Lennox at 37 °C. Selective drug concentrations for E. coli were 50 µg ampicillin ml–1, 34 µg chloramphenicol ml–1 and 25 µg kanamycin ml–1.
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; Narumi et al., 2001; Nishida & Narumi, 2002). The previously constructed disruption plasmid pKSCR3 (carrying the recA229 : : cat mutation) (Narumi et al., 2001) was used to disrupt the recA gene. To construct plasmids carrying the lexA1 null mutation, a 975 bp HincII fragment (KatAPH cassette) from pKatAPH3 (Ohba et al., 2005) or a 948 bp HincII fragment (KatAAD cassette) from pKatAAD2, which is a spectinomycin-resistant version of pKatAPH3, was ligated to EagI-digested pZA8 (Kikuchi et al., 1999). The resulting plasmids were designated pLKN2 (carrying the lexA1-166 : : aph mutation) and pLAN2 (carrying the lexA1-166 : : aad mutation), respectively.
pDC201 is a cosmid clone of the genome library (Narumi et al., 1997), and it was used to clone the D. radiodurans lexA2 gene. The 5.6 kb EcoRI fragment carrying the D. radiodurans lexA2 locus was isolated from pDC201 and subcloned in the EcoRI site of pUC19. The resulting plasmid was designated pLE44. Plasmid pLE44 was digested with BsrGI and HindIII, blunted with T4 DNA polymerase, and then self-ligated to yield plasmid pLEB7. Plasmid pLEB7 was subjected to site-directed mutagenesis to create a unique SmaI site in lexA2 (T at nucleotide position 365 was changed to G). Site-directed mutagenesis was carried out using the QuikChange Site-directed Mutagenesis kit (Stratagene) with primers T365G-F (5'-GTGCAGCAGGTCCCGGGCTACATCGCCGGTCAG-3') and T365G-R (5'-CTGACCGGCGATGTAGCCCGGGACCTGCTGCAC-3'), and the resulting plasmid was designated pLSM2. The KatAPH cassette was ligated to SmaI-digested pLSM2 to construct plasmids carrying the lexA2 null mutation, and the resulting plasmid was designated pLK2N2 (carrying the lexA2-362 : : aph mutation).
All disruptant strains were generated from D. radiodurans R1 (ATCC 13939). The recA229 : : cat, lexA1-166 : : aph and lexA2-362 : : aph mutations were introduced into the genome of D. radiodurans and the resulting disruptant strains were designated XRA1 (recA disruptant), XLK1 (lexA1 disruptant) and XL2K1 (lexA2 disruptant), respectively. We introduced the lexA1 null mutation into the recA disruptant strain and the resulting recA lexA1 double-disruptant strain was designated XRL11. Additionally, the recA null mutation was introduced into the lexA2 disruptant strain to yield the recA lexA2 double-disruptant strain XL2R8, and the lexA1 null mutation was introduced into the lexA2 disruptant strain to yield the lexA2 lexA1 double-disruptant strain WXL1.
Construction of expression plasmid and DNA sequencing of the D. radiodurans lexA2 gene.
To isolate the lexA2 coding region, PCR was carried out using PfuTurbo HotStart DNA polymerase (Stratagene) with pLEB7 and specific oligonucleotides A74EX-F3 (5'-CTCAGTCTAATTCGCATATGCGAACATTTC-3') and A74EX-R2 (5'-GCAGGGATCCGGGCAGAGGCTCAGGC-3'), possessing NdeI and BamHI restriction sites (underlined in the sequences), respectively. PCR products were then digested with NdeI and BamHI to adapt the termini for in-frame insertion of lexA2 into the NdeI–BamHI sites in the pET3a vector (Novagen). The resulting expression plasmid was designated pET3lexA2. The DNA sequence of the expression plasmids was checked to confirm the absence of errors by PCR using T7 promoter primer (5'-TAATACGACTCACTATAGGG-3') and T7 terminator primer (5'-GCTAGTTATTGCTCAGCGG-3').
To check the sequence of the lexA2 locus in plasmid pLEB7 and the D. radiodurans genome, direct sequencing was carried out using the oligonucleotide primer sets A74DI-F (5'-ATGGCCTGATCGCAGAACGTATG-3') and A74DI-R (5'-TGTCACTCCCTGAAAGGTGCTAGA-3').
RNA isolation and primer extension assay.
D. radiodurans cells were grown in TGY broth at 30 °C with agitation to early stationary phase. Total RNA was isolated from the harvested cells using a FastRNA Pro Blue Kit (Qbiogene) with a DNA-free kit (Ambion). The specific oligonucleotide IRD800lexA2 (5'-CCTTCGCCGCGCTGGTAGCAAG-3') was used to map the transcriptional start site of the lexA2 mRNA. RNA (10 µg) and the IRD800 infrared dye-labelled primer (1 pmol) were incubated in a buffer containing 10 mM Tris/HCl (pH 7.4), 1 mM EDTA and 250 mM KCl at 64 °C for 1 h. After annealing, reverse transcription was carried out using Superscript III RNase H– reverse transcriptase (Invitrogen) as recommended by the manufacturer. Analysis of the primer extension product was performed using a DNA Analyser model 4200-1G (LI-COR). The size of the primer extension product was determined by comparison with the DNA sequence generated using the same primer with plasmid pLEB7 as a template.
Protein purification.
E. coli BLR(DE3) carrying pET3lexA2 was cultivated in LB broth-Miller containing ampicillin. At an OD600 of approximately 0.5, IPTG was added to a final concentration of 0.4 mM and growth was continued for an additional 2 h. The cells were then harvested by centrifugation. The cell pellet was resuspended in cold buffer P1 (20 mM sodium phosphate pH 6.0 and 0.1 mM EDTA) containing 1 mM PMSF and 0.1 % (w/v) protease inhibitor cocktail (Calbiochem) in 1/100 of the original culture volume, and stored at –80 °C. All subsequent steps were carried out at 4 °C unless otherwise indicated.
Cells were allowed to thaw with occasional stirring and the suspension was sonicated for 10 min on ice; debris was removed by centrifugation at 10 000 g, 4 °C for 30 min. The supernatant was then applied to a HiPrep 16/10 Heparin FF column (GE Healthcare) equilibrated with buffer P1. The column was washed with four column volumes of buffer P1. Following elution with 10 volumes of a linear NaCl gradient (0 to 1 M), the fractions containing LexA2 were pooled. The majority of LexA2 was eluted at approximately 0.6 M NaCl. The pooled fractions were concentrated and desalted using an Amicon Ultra-15 filter unit (Millipore) with buffer P1, and the fraction was then loaded onto a Mono S HR 5/5 column (GE Healthcare) equilibrated with buffer P1. The column was washed with 10 column volumes of buffer P1. Following elution with 20 volumes of a linear NaCl gradient (0.3 to 1 M), the fractions containing LexA2 were pooled. The majority of LexA2 was eluted at approximately 0.5 M NaCl. The pooled fractions were concentrated using an Amicon Ultra-15 filter unit with buffer P1.
Autodigestion and RecA-mediated cleavage reaction.
The autodigestion reaction was assayed by incubating 8 µM LexA2 in 50 mM Tris/HCl (pH 10.0) at 37 °C for 16 h. For the cleavage of LexA2 mediated by D. radiodurans RecA, the reaction mixture contained 20 mM Tris/HCl (pH 6.0), 10 mM MgCl2, 6 µM oligonucleotide (5'-GAGAACCATGGATGGACCAATAATAATGACTAGAG-3'; 35-mer), 1 mM ATPS, 4.2 µM RecA (Satoh et al., 2002) and 8 µM LexA2. To activate RecA by binding with ATPS and oligonucleotide, the mixture without LexA2 was first combined in a microtube and preincubated at 37 °C for 10 min. The reaction was then initiated by addition of LexA2, and the reaction mixture was incubated at 37 °C for 1 h. After quenching by addition to SDS sample buffer and heating at 95 °C for 10 min, the reaction products were subjected to 15 % (w/v) SDS-PAGE. LexA2 and breakdown products were visualized by staining with GelCode Blue Stain Reagent (Pierce).
Detection of intracellular levels of D. radiodurans RecA, LexA1 and LexA2.
D. radiodurans cells were grown in TGY broth at 30 °C with agitation to early stationary phase. Cells were harvested by centrifugation at 5000 g, 4 °C for 5 min, washed twice with 10 mM sodium phosphate buffer (pH 7.0) and resuspended in the same buffer. Aliquots (20 ml) of the cell suspension were irradiated at room temperature for 1 h with 60Co -rays at a dose rate of 2 kGy h–1. The cells were then centrifuged at 5000 g, 4 °C for 5 min and resuspended in 200 ml TGY broth. The cell suspension was incubated at 30 °C for different post-irradiation times (0, 0.5, 1 and 2 h). Aliquots (80 ml) of the culture were then harvested as needed. To obtain crude protein extract, the cells were resuspended in lysis buffer (0.5 ml) containing 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, 1 mM PMSF, 0.1 % (w/v) protease inhibitor cocktail (Calbiochem) and 1 % (w/v) SDS, and disrupted using a FastPrep Cell Disruptor FP120 (Savant Instruments) with a FastProtein Blue Kit (Qbiogene). Following removal of cell debris by centrifugation, the supernatant was subjected to 12.5 or 15 % (w/v) SDS-PAGE. The resolved proteins were transferred onto a PVDF membrane (Millipore). D. radiodurans RecA, LexA1 or LexA2 were detected using D. radiodurans RecA antibody (diluted 1 : 10 000), D. radiodurans LexA1 antibody (diluted 1 : 10 000) (Narumi et al., 2001) or D. radiodurans LexA2 antibody (diluted 1 : 4000) together with alkaline-phosphatase-conjugated anti-mouse or anti-rabbit IgG antisera (Applied Biosystems). The D. radiodurans RecA and LexA2 antibodies (mouse, polyclonal) were raised against purified D. radiodurans RecA (Satoh et al., 2002) and purified D. radiodurans LexA2, respectively, at Immuno-Biological Laboratories (Gunma, Japan). Chemiluminescent signals on the PVDF membrane were visualized using a Lumi-Imager F1 Workstation (Roche Diagnostics). D. radiodurans LexA1 and LexA2 antibodies did not cross-react with each antigen.
Measurement of cell survival rate.
D. radiodurans cells were grown, harvested, washed and resuspended as described above. Aliquots (0.1 ml) of the cell suspension were dispensed into test tubes and irradiated at room temperature for 1 to 3 h with 60Co -rays at dose rates from 0.1 to 5 kGy h–1 that were regulated by changing the distance of the samples from the -ray source. Irradiated samples were appropriately diluted with 10 mM phosphate buffer, spread onto TGY agar, and incubated at 30 °C for 3 days prior to the enumeration of colonies.
Luciferase reporter assay.
The luciferase reporter plasmid pHOB12R contained engineered firefly luciferase gene (FL), Renilla reniformis luciferase gene (RL) and the chloramphenicol resistance gene as a genetic marker. The expression of FL and RL was regulated by the D. radiodurans pprA and groE minimal promoters, respectively (Ohba et al., 2005). The luciferase reporter plasmid p12R-Hyg, which carries a hygromycin-resistance gene instead of a chloramphenicol-resistance gene, was constructed as a pHOB12R-based plasmid. A 1199 bp HincII fragment (KatHPH cassette) from pKatHPH4, which is a hygromycin-resistant version of pKatAPH3, was ligated to MscI- and FspI-digested pHOB12R. D. radiodurans cells carrying the resulting plasmid p12R-Hyg were grown, harvested, washed and resuspended as described above. Aliquots (1 ml) of the cell suspension were irradiated at room temperature for 2 h with 60Co -rays at a dose rate of 4 kGy h–1. The cells were then centrifuged at 5000 g, 4 °C for 5 min, and resuspended in 4 ml TGY broth. The cell suspension was incubated at 30 °C for different post-irradiation incubation times, with agitation. FL and RL activities in cells (20 µl) were measured using the Dual-Glo Luciferase Assay System (Promega). Chemiluminescent signals were visualized using a Lumi-Imager F1 Workstation. The relative reporter activity was calculated as the ratio of the luminescence from FL (experimental reporter) to the luminescence from RL (control reporter).
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RESULTS |
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) that was not present in the DRA0074 sequence. Furthermore, the lexA2 loci in D. radiodurans strains MR1, KR1 and R1, which are different isolates of the wild-type strain (Islam et al., 2003), were resequenced using primers A74DI-F and A74DI-R. In each case, the sequence was identical to our lexA2 sequence, suggesting an error in the previous sequence. We deposited our lexA2 sequence in GenBank under accession no. AB256032. The addition of one nucleotide affected the open reading frame; 44 amino acid residues were altered, and the predicted amino acid sequence of lexA2 was 8 amino acid residues longer than the previous sequence (Fig. 1). Fig. 2 shows the alignment of the amino acid sequence of E. coli LexA and D. radiodurans LexA1 and LexA2. Although D. radiodurans LexA2 exhibited low similarity to LexA of E. coli and D. radiodurans (23.4 % and 24.4 % identity, respectively), LexA2 retained the amino acid residues involved in self-cleavage of the protein (Ala-130, Gly-131, Ser-163 and Lys-203) and a helix–turn–helix DNA-binding motif (Ile-38 to Leu-100) (Fig. 2). The C-terminal region of E. coli LexA (Phe-186 to Arg-197) participates in dimeric interactions (Fig. 2) (Luo et al., 2001). The C-terminal region of LexA2 revealed in this study may also be involved in dimeric interactions in a manner similar to E. coli LexA. The amino acid sequence alignment shows that D. radiodurans LexA2 has a longer N-terminal sequence than E. coli and D. radiodurans LexA1 proteins (Fig. 2). The primer extension assay revealed that the transcription start site of lexA2 was located at –97 and –98 upstream of the translation start site (Fig. 3). No promoter-like sequence was found between the translation start and transcription start sites for lexA2. These results support the existence of an extended N-terminal region in D. radiodurans LexA2.
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). This was very close to the molecular mass (26 750 Da) calculated from the amino acid sequence of LexA2.
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). Similar to E. coli LexA, D. radiodurans LexA1 undergoes autodigestion when subjected to alkaline conditions and RecA-mediated cleavage (Narumi et al., 2001). We investigated whether these proteolytic cleavage properties are conserved in D. radiodurans LexA2. Purified LexA2 was autodigested at pH 10 to yield two breakdown products (Fig. 4b, lane 2). LexA2 was cleaved by incubation with D. radiodurans RecA to produce two breakdown products whose sizes are the same as those observed in autodigestion (Fig. 4b, lane 6). No breakdown product was observed when RecA, oligonucleotide or ATPS was omitted from the reaction (Fig. 4b, lanes 3 to 5). These results support the conclusion that D. radiodurans LexA2 maintains proteolytic activity that can be stimulated by D. radiodurans RecA.
Changes in intracellular RecA, LexA1 and LexA2 levels in disruptants following irradiation
To determine if LexA2 is involved in RecA induction and whether cleavage of LexA2 is promoted by RecA in vivo, changes in RecA, LexA1 and LexA2 levels following -irradiation (2 kGy) were compared between parental and disruptant strains.
Fig. 5 shows the results of experiments in which cells treated with or without radiation were incubated in fresh TGY broth for different post-irradiation incubation times and then subjected to Western blot analysis using D. radiodurans RecA, LexA1 or LexA2 antibodies. The intracellular level of RecA increased in the lexA1 disruptant strain following -irradiation as in the wild-type strain (Fig. 5a). The LexA1 level in the recA disruptant strain did not change for any of the post-irradiation incubation times examined (Fig. 5b). These results confirmed our previous conclusions that D. radiodurans LexA1 is not involved in RecA induction following -irradiation and that RecA is the sole protein required for LexA1 cleavage (Narumi et al., 2001). RecA induction following -irradiation was also observed in the lexA2 disruptant and lexA1 lexA2 double-disruptant strains (Fig. 5a), clearly indicating that neither LexA1 nor LexA2 is involved in RecA induction. While a decreased level of LexA2 was observed in wild-type and lexA1 disruptant strains following -irradiation for all post-irradiation incubation times (Fig. 5c), -irradiation did not affect the level of LexA2 in the recA disruptant strain. This result indicated that RecA is the sole protein required for LexA2 cleavage, and supported our observation of in vitro RecA-mediated LexA2 cleavage (Fig. 4b).
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). Surprisingly, the LexA1 level in the lexA2 disruptant strain almost recovered 1 h after irradiation, although the decrease in the LexA1 level was observed until 0.5 h in the case of the wild-type strain (Fig. 5b). The damaged DNA leads to the production of segments of ssDNA that act as a substrate for RecA activation. As in E. coli, the co-protease of D. radiodurans RecA could not activate in the absence of ssDNA (Fig. 4b, lane 4). The segments of ssDNA disappear as a consequence of various DNA repair processes. Since the decrease in the intracellular LexA1 level reflects the co-protease activity of RecA, the recovery to a basal level of LexA1 indicates the inactivation of RecA (Fig. 5b). This result suggested that the inactivation of RecA was faster in the lexA2 disruptant strain than in the wild-type strain. Given that the inactivation of RecA is associated with the completion of DNA repair, the DNA repair capacity might be enhanced in the lexA2 disruptant strain relative to the wild-type strain.
Sensitivity of gene disruptant strains to -rays
To investigate DNA repair capacities, we examined the sensitivity of the disruptant strains to -rays. As shown in Fig. 6(a), the lexA2 disruptant and lexA1 lexA2 double-disruptant strains exhibited a much higher resistance than the wild-type strain at high doses (8 to 15 kGy) of -rays. The LD90 values (12.7 kGy) of the lexA2 disruptant and lexA1 lexA2 double-disruptant strains were 1.6-fold higher than those for the wild-type strain (7.8 kGy). On the other hand, the radioresistance of the lexA1 disruptant strain was equal to that of the wild-type strain.
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, the recA disruptant strain exhibited extreme sensitivity (LD90 value 0.09 kGy). In a recA background, neither lexA1 nor lexA2 disruption altered the radiosensitivity of D. radiodurans (LD90 values 0.09 and 0.12 kGy, respectively).
Change in pprA promoter activation in disruptants following -irradiation
The pprA gene encoding the novel radiation-inducible protein PprA plays a critical role in the radioresistance of D. radiodurans (Narumi et al., 2004). To investigate whether LexA homologues are involved in PprA induction, changes in pprA promoter activation following -irradiation (8 kGy) were compared between parental and disruptant strains using the luciferase reporter assay. pprA promoter activation reached a maximum at 3 h post-irradiation incubation in all of the strains (Fig. 7). Remarkably, the maximum level of pprA promoter activation in the lexA2 disruptant strain was approximately 1.4-fold higher than that recorded for the wild-type strain. The change in pprA promoter activation in the lexA1 disruptant strain was very similar to that of the wild-type strain. On the other hand, the maximum level of pprA promoter activation in recA disruptant strains was lower than that of the wild-type strain. The maximum level of pprA promoter activation of the recA lexA2 double-disruptant strain was slightly higher than that recorded for the recA disruptant strain. These results indicated that the pprA promoter activation was enhanced by lexA2 disruption.
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) and found a single base difference between our sequence and the published D. radiodurans R1 genome sequence (White et al., 1999). It was reported recently that the D. radiodurans strain used in the genome project was not the R1 type strain that had been deposited in the American Type Culture Collection as ATCC no. 13939, but that it was the strain designated BAA-816 in which it was suspected many mutations were acquired during years of separate culture (Mennecier et al., 2004). Although resequencing of the lexA2 locus in this study revealed the correct sequence, the fact remains that D. radiodurans contained a second, diverged copy of lexA1.
We purified D. radiodurans LexA2 to obtain a molecular mass of approximately 26 kDa (Fig. 4a), which was smaller than the molecular mass (approx. 33 kDa) of D. radiodurans DRA0074 purified by Sheng et al. (2004). For this reason it was thought that the lexA2 stop codon of the expression plasmid pET3lexA2wt constructed in this study differed from the expression plasmid constructed by Sheng et al. (2004) because they had designed PCR primers for amplifying the dra0074 gene on the basis of the published sequence (White et al., 1999). Consequently, the DRA0074 protein purified by Sheng et al. (2004) may contain amino acid residues that originated from a vector at its C-terminal region.
The N-terminal region of E. coli LexA is involved in DNA-binding activity (Fogh et al., 1994). The purification of D. radiodurans LexA2 showed a heparin column to be very effective (Fig. 4a), suggesting that LexA2 retains DNA-binding ability. Amino acid sequence alignment revealed that the N-terminal region of D. radiodurans LexA2 did not possess similarities to the E. coli and D. radiodurans LexA1 proteins (Fig. 2). These results suggested that D. radiodurans possesses distinct LexA1- and LexA2-binding motifs that differ from the E. coli SOS box (Walker, 1984; Friedberg et al., 1995). D. radiodurans LexA1 and LexA2 may be involved in different gene regulations.
A hypothesis concerning the radiation response mechanism in D. radiodurans is presented in Fig. 8 on the basis of the results obtained in the present and previous studies. Unlike E. coli, D. radiodurans LexA1 is not involved in RecA induction following -irradiation (Narumi et al., 2001; Bonacossa de Almeida et al., 2002). Furthermore, neither the lexA1 gene deletion nor the mutation leading to a non-cleavable LexA1 affected survival or double-strand breaks repair, suggesting that LexA1 does not play a major role in the radiation response mechanism following -irradiation in D. radiodurans (Jolivet et al., 2005). The results of radioresistance in the lexA1 disruptant strains (Fig. 6) are consistent with these conclusions. A previous study assumed that LexA1 and DRA0074 have similar regulatory functions in the metabolic pathway because a lexA1 dra0074 double-disruptant strain could not be constructed (Sheng et al., 2004). However, we successfully constructed a lexA1 lexA2 double-disruptant strain in the present study. The induction of RecA was observed in the lexA1 lexA2 double-disruptant strain (Fig. 5a), indicating that the two LexA homologues do not exhibit functional overlap in relation to RecA induction. These results support the notion that recA expression following -irradiation is regulated by DR0167 (also called PprI/IrrE) in D. radiodurans (Earl et al., 2002; Hua et al., 2003) (Fig. 8).
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-rays compared with the wild-type (Satoh et al., 2002). RecA can accelerate autocleavage of D. radiodurans LexA1 (Narumi et al., 2001), and LexA2 autocleavage is also accelerated by RecA (Fig. 4b, lane 6). This suggests that a certain gene down-regulated by LexA1 and/or LexA2 might be important for the radioresistance of D. radiodurans.
Sheng et al. (2004) examined the radiosensitivity of dra0074 disruptant strain M74 up to a dose of 8 kGy, and found no significant difference in survival curves between strain M74 and the wild-type. However, we found that lexA2 disruptant strains exhibited a much higher resistance to -rays than the wild-type at high doses (8 to 15 kGy) (Fig. 6a). At least part of the increase in radioresistance of the lexA2 disruptant strain may be explained by the enhancement of pprA promoter activation (Fig. 8). D. radiodurans PprA preferentially binds to dsDNA carrying strand breaks (Narumi et al., 2004). DNA strand breaks also result from the process of DNA replication. We suggest that the intracellular PprA level must be regulated. If PprA is induced constitutively, cell growth will be inhibited in D. radiodurans because PprA binds to DNA strand breaks that are generated by DNA replication. Therefore, the regulation by LexA2 contributes to the stable maintenance of cell growth in the undamaged state. While an enhancement of pprA promoter activation in the recA lexA2 double-disruptant strain was not prominent (Fig. 7), lexA2 disruption did not restore radiosensitivity in the recA background (Fig. 6b). This finding suggested that the radiation response mechanism proceeds in a RecA-dependent manner.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 16 May 2006;
revised 3 August 2006;
accepted 17 August 2006.
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-helices (H1, H2 and H3) found in E. coli LexA are shown according to the description of Fogh et al. (1994)
, Wild-type; 
, recA disruptant strain; 
, lexA1 disruptant strain; 
, lexA2 disruptant strain; 
, lexA1 lexA2 double-disruptant strain.
, recA lexA1 double-disruptant strain; 
, recA lexA2 double-disruptant strain.
