Ppra Research Paper

Introduction

Deinococcus radiodurans possesses extraordinary resistance to the lethal and mutagenic effects of ionizing and UV radiation and many other DNA damaging agents, which is thought to be effected by a highly proficient DNA repair capacity (Moseley, 1983; Battista, 1997). The most striking feature of D. radiodurans is its capacity for repairing ionizing radiation-induced DNA double-strand breaks (DSBs). This bacterium can mend over 100 DSBs of genomic DNA during post-irradiation incubation, whereas just a few DSBs are lethal in nearly all other living organisms (Dean et al., 1966). Because the D. radiodurans genome is polyploid under the conditions of growth used in the laboratory (Hansen, 1978; Harsojo et al., 1981), repair of the irradiation-induced DNA DSBs is thought to occur mainly through homologous recombination (Minton, 1994). This highly efficient DNA DSB repair process is radiation inducible and is dependent on de novo protein synthesis following irradiation (Kitayama and Matsuyama, 1968). Recent transcriptome analysis has revealed that many genes implicated in DNA repair, recombination and replication, in addition to several uncharacterized genes, exhibited a recA-like activation pattern during recovery after exposure to ionizing radiation (Liu et al., 2003). The RecA recombinase protein is believed to play a critical role in the homologous recombination repair of radiation-induced DNA DSBs in D. radiodurans (Minton, 1994). Because the D. radiodurans RecA protein binds much better to double-stranded DNA (dsDNA) than its Escherichia coli counterpart and promotes DNA strand exchange via a pathway that is the inverse of that catalysed by the E. coli RecA protein, it was proposed that this peculiar characteristic of RecA may reflect the remarkably efficient system for DNA DSB repair in D. radiodurans (Kim and Cox, 2002; Haruta et al., 2003). On the one hand, molecular genetic and biochemical studies with D. radiodurans RecA mutants suggested that radiation resistance through RecA recombination activity plays less of a role than its co-protease activity (Satoh et al., 2002). This implies that D. radiodurans possesses a RecA-independent recombination mechanism and/or a DNA repair mechanism other than homologous recombination. Because recA-deficient D. radiodurans was able to rejoin many DSBs in a sequence-specific manner at early times after irradiation, it was proposed that the earliest phase of DNA repair is dominated by a recA-independent single-strand annealing reaction (Daly and Minton, 1996). However, a gene involved in DSB repair before recA-dependent recombination has yet to be identified. The debate concerning whether D. radiodurans uses a DNA repair system that includes novel components has received much attention in recent years (Edwards and Battista, 2003; Narumi, 2003).

Over 40 natural mutant strains have been isolated in our investigations dealing with DNA damage repair-deficient mutants of D. radiodurans. Strain KH311 exhibited significant sensitivity to γ-rays, mitomycin-C (MMC), UV radiation and 4,5′,8-trimethylpsoralen (Kitayama et al., 1983). In this report, we described the identification of a unique radiation-inducible gene (DRA0346, designated pprA) responsible for loss of radiation resistance from this mutant. To gain insight into the nature of the gene product (PprA) in D. radiodurans, the protein was purified and characterized. Our results suggest that D. radiodurans has a radiation-induced non-homologous end-joining (NHEJ) repair mechanism in which PprA plays a critical role.

Results and discussion

Identification of pprA

In an effort to identify the gene responsible for DNA repair deficiency of the mutant strain KH311 (Kitayama et al., 1983), the DNA of a cosmid library from the radiation-resistant strain (Narumi et al., 1997) was introduced into the mutant, and clones capable of reverting the mutant to acquire resistance to MMC were screened. We found that the MMC sensitivity of KH311 was restored by transforming with DNA derived from a cosmid clone (Fig. 1). In an effort to identify the defective locus in KH311, a deletion library of the cosmid was examined for transforming activity. As a result, the defective locus was localized within a 2.9 kb region. Sequence comparison of this region between wild type and KH311 revealed that the mutation site in strain KH311 was identified as a G:C to A:T transition at nucleotide position 446 in DRA0346 (White et al., 1999). We designated DRA0346 as a pleiotropic protein promoting DNA repair (pprA). The amino acid sequence deduced from pprA showed no homology to other known proteins. Mutation of pprA in KH311 (pprA446) results in an amino acid substitution at position 149 (Gly to Glu) of the gene product. To further investigate the physiological function of the gene, we generated a pprA disruptant strain (XN1 carrying pprA428::cat) in a wild-type background using a direct insertional mutagenesis technique (Funayama et al., 1999). The sensitivity of XN1 to γ-rays was the same as KH311 (Fig. 2), indicating that the pprA446 mutation in KH311 resulted in a complete loss of function of the gene.

Relationship between PprA and RecA in DNA repair

Transcriptome analysis has demonstrated that DRA0346 (pprA) exhibits a recA-like activation pattern during recovery from exposure to ionizing radiation (Liu et al., 2003). Recent work on another DNA damage repair-deficient mutant of D. radiodurans identified a novel regulatory protein (DR0167) that stimulated transcription of the recA gene after γ-irradiation (Earl et al., 2002). It has been reported that the radiation response of pprA, in addition to recA, was eliminated in the DR0167 disruptant strain (Hua et al., 2003). In an effort to investigate the relationship between pprA and recA in DNA repair, we generated a pprA disruptant in a recA background (KS2 carrying pprA428::cat and recA424). The KS2 double mutant was more sensitive to γ-rays than the pprA single mutant or the pprA disruptant strains (Fig. 2). It has been shown that RecA424 did not promote DNA strand exchange reaction under conditions in which the wild-type RecA could promote the reaction when ATP was used as a nucleotide cofactor. In the presence of dATP that is a more effective nucleotide cofactor than ATP, RecA424 could convert substrate DNA to strand exchange intermediates to a certain extent. However, no complete strand exchange product was observed with RecA424 (Satoh et al., 2002). Therefore, there are two possibilities to explain the increased sensitivity to γ-rays of the KS2 double mutant: (i) PprA promotes the strand exchange reaction by RecA424 or (ii) PprA plays an important role in radiation resistance in a RecA-independent manner and is involved in the single-strand annealing pathway (Daly and Minton, 1996). In our view, the latter was more likely. To test our hypothesis, we tried to generate a pprA and recA double knockout strain. Despite repeated attempts, we failed to obtain the double knockout strain. Perhaps the double lack of pprA and recA is lethal for D. radiodurans cell.

With a view to investigate the physiological relationship between PprA and RecA at the protein level, PprA protein was purified from recombinant E. coli(Fig. 3), and PprA anti-serum was generated using the purified recombinant protein. We examined changes in the intracellular levels of PprA and RecA after γ-irradiation. As shown in Fig. 4, induction of RecA was not affected by the pprA mutation or disruption, nor was the induction of PprA affected by the recA disruption, confirming that PprA is important for DNA repair. These findings support the notion that DR0167 plays a critical role in regulating multiple DNA repair pathways in response to radiation stress in D. radiodurans. The genome sequence of Thermus thermophilus HB27, which represents the most closely related genus to Deinococcus, was recently published (Henne et al., 2004). Using these data, we searched for pprA and DR0167 homologues in the T. thermophilus genome but were unable to find either a pprA or a DR0167 homologue, highlighting the uniqueness of the DNA repair and DNA damage response mechanisms of D. radiodurans.

DNA binding property of PprA protein

We found a difference in the agarose gel DNA migration pattern between the crude protein extracts from the recombinant E. coli producing wild-type PprA and mutant PprA446 (a single amino acid substitution; Gly149Glu) (Fig. 5A and B). When the crude protein extract was mixed with linear plasmid dsDNA, the difference in the DNA migration pattern was even more marked. These results indicated that wild-type PprA possesses DNA binding ability whereas the PprA446 mutant loses DNA binding ability. The purified wild-type PprA protein was then closely evaluated for DNA binding ability. Agarose gel electrophoretic mobility-shift assay using φX174 DNA demonstrated that PprA formed a specific complex with either dsDNA in the open circular form (RF II) or linear dsDNA, but did not bind to either dsDNA in the closed circular form (RF I) or single-stranded DNA (ssDNA) (Fig. 5C). This indicated that PprA binds preferentially to dsDNA carrying strand breaks, suggesting a role for PprA in DNA repair. Furthermore, under conditions where a substantial amount of degradation of linear dsDNA would normally result as a result of E. coli exonuclease III activity, no DNA degradation was observed when linear dsDNA was pre-incubated with PprA (Fig. 5D). These results support the notion that PprA binds to dsDNA carrying strand breaks, which could play a role in shielding radiation-induced DNA DSBs from exonuclease activity and subsequent degradation, thereby facilitating unabated DNA repair processes.

The aforementioned property of PprA reflects the DNA binding properties of eukaryotic Ku (Bliss and Lane, 1997) and Rad50/Mre11/Xrs2 (Chen et al., 2001), both of which are involved in NHEJ. Eukaryotic Ku binds to dsDNA ends with high affinity (Bliss and Lane, 1997). The yeast Rad50/Mre11/Xrs2 complex juxtaposes linear dsDNA via their ends to form oligomers (Chen et al., 2001). Additionally, the Rad50/Mre11/Xrs2 complex protects dsDNA from digestion by λ-exonuclease and E. coli exonuclease III (Chen et al., 2001). Recently, bacterial Ku homologues were identified from Bacillus subtilis (YkoV) and Mycobacterium tuberculosis (Rv0937c). Disruption of YkoV rendered B. subtilis more sensitive to ionizing radiation, and Rv0937 preferentially bound to dsDNA ends (Weller et al., 2002). Although PprA possesses no homology to eukaryotic Ku, nor to either component of the yeast Rad50/Mre11/Xrs2 complex or bacterial Ku homologues, it is tempting to speculate that D. radiodurans possesses an NHEJ pathway for the repair of radiation-induced DNA DSBs in which PprA plays a critical role.

PprA stimulates DNA ligase-catalysed DNA end-joining

DNA-bound eukaryotic Ku acts by recruiting the IV/XRCC4 ligase complex, thereby facilitating DNA ligation (Kysela et al., 2003). The yeast Rad50/Mre11/Xrs2 complex directly interacts with and specifically stimulates Dnl4/Lif1-mediated intermolecular ligation (Chen et al., 2001; Trujillo et al., 2003). The M. tuberculosis Ku homologue (Rv0937c) stimulates the ligation activity of a genetically linked ATP-dependent DNA ligase (Rv0938) (Weller et al., 2002). Therefore, we became interested in determining whether PprA could stimulate the ligation activity of DNA ligase. As shown in Fig. 6A, PprA stimulated DNA ligation reactions catalysed by T4 DNA ligase and E. coli DNA ligase at a concentration resulting in the complete inhibition of E. coli exonuclease activity, although PprA itself did not possess DNA ligation activity. The degree of stimulation was dependent on the concentration of PprA when the concentration of DNA substrate and DNA ligase remained constant (Fig. 6B). At the optimum PprA concentration, the percentage of ligated products relative to the total amount of DNA was 56% and 26% in the reactions containing T4 DNA ligase and E. coli DNA ligase respectively. Much higher concentrations of PprA resulted in the generation of fewer ligation products. These results indicated that PprA stimulated the DNA ligase-catalysed DNA end-joining reaction as in the case of Ku and Rad50/Mre11/Xrs2, supporting the notion that D. radiodurans possesses an NHEJ pathway for the repair of radiation-induced DNA DSBs. It should be noted that this type of pathway may be error prone because DNA end-produced by irradiation probably undergo clustered damages, the removal of which can result in mutation. Therefore, the NHEJ pathway must be accompanied by a mechanism that prevents mutation to achieve accurate DNA DSB repair in D. radiodurans (Narumi, 2003). This point requires clarification in an effort to better understand the mechanisms involved in DNA repair.

Yeast Rad50/Mre11/Xrs2 complex- and M. tuberculosis Ku homologue-mediated stimulations of ligation are highly specific for ligases from the same species (Chen et al., 2001; Weller et al., 2002). D. radiodurans has a typical bacterial NAD-dependent DNA ligase (DR2069) and a diverged homologue of ATP-dependent DNA ligase (DRB0100) that possesses similarity to eukaryotic DNA ligase III. After γ-irradiation, the former was repressed, whereas the latter exhibited a recA-like activation pattern (Liu et al., 2003). Because pprA exhibits a recA-like activation pattern, it is a tantalizing possibility that DRB0100 is a counterpart of PprA in the radiation-induced NHEJ pathway in D. radiodurans. Further experiments, in which DRB0100 is assayed for DNA ligation activity, will be required to address this question.

Although T4 DNA ligase was not stimulated by either yeast Rad50/Mre11/Xrs2 or the M. tuberculosis Ku homologue (Chen et al., 2001; Weller et al., 2002), PprA was able to stimulate the ligation activity of T4 DNA ligase. Given that T4 DNA ligase is widely used in DNA engineering, the possibility of making use of PprA in combination with T4 DNA ligase was examined. We found that the cloning efficiency of a commercially available TA vector with T4 DNA ligase was enhanced by more than twofold after the addition of an appropriate concentration of PprA to the reaction mixture (Fig. 6C). Thus, the properties of PprA, which include recognition of and binding to DNA ends and the stimulation of DNA ligation, suggest its potential use as a reagent in DNA engineering.

Experimental procedures

Growth, radiation treatment and survival curves of D. radiodurans

All D. radiodurans strains used in this work were grown at 30°C in TGY broth (0.5% Bacto Tryptone, 0.3% Bacto Yeast Extract, 0.1% glucose) or on TGY agar supplemented with 1.5% Bacto Agar. Chloramphenicol at 3 µg ml−1 was supplemented if necessary. D. radiodurans cells were grown to early stationary phase and used for determining the cell survival rate. For irradiation with γ-rays, the cell suspension was irradiated with 60Co γ-rays at several different doses. After treatment, cells were diluted in 10 mM phosphate buffer, plated on TGY agars and incubated at 30°C for 3 days before the colonies being enumerated.

Disruption of pprA

Disruption of pprA was achieved using a previously described direct insertional mutagenesis technique (Funayama et al., 1999) with modifications. Briefly, a 6.0 kb DNA fragment containing pprA was obtained from the cosmid pDC144 through restricted digestion by SalI and SacI (Narumi et al., 2001). This was then ligated into pUC19, pre-digested with SalI and SacI, to yield plasmid pZA8. Because the pprA coding region possesses a single NspV site at the middle, the plasmid was digested with NspV, blunted with T4 DNA polymerase and then ligated to a 0.9 kb HincII fragment containing the chloramphenicol resistance gene (cat) from pKatCAT (Funayama et al., 1999). This disruption plasmid was linearized by ScaI and used to transform D. radiodurans KD8301 (wild type) (Narumi et al., 1997) or KI696 (recA424) (Satoh et al., 2002). Chloramphenicol-resistant colonies were collected, and disruption of the pprA gene was confirmed by amplifying the targeted allele by genomic polymerase chain reaction (PCR) (Funayama et al., 1999; Hua et al., 2003). Strains possessing the pprA disruption by insertional mutagenesis were designated XN1 (carrying pprA428::cat) and KS2 (carrying pprA428::cat recA424).

Expression plasmid construction

In an effort to isolate the pprA coding region, PCR was carried out using pZA8 DNA with the specific oligonucleotides 5′-GGGCATAATAAAGGCCATATGGCAAGGCTAAA GC-3′ and 5′-TTTTGGATCCTCAGCTCTCGCGCAGGCCG TGC-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 pprA into the NdeI-BamHI sites in the pET3a vector (Novagen). The resultant expression plasmid was designated pET3pprAwt. Introduction of the pprA446 mutation in pET3pprAwt was performed using a QuikChange Site-Directed Mutagenesis Kit (Stratagene), and the resultant plasmid was designated pET3pprA446. The DNA sequences of the expression plasmids were checked to confirm the absence of any errors that may have been introduced by PCR. DNA sequencing was performed using an ABI PRISM 377XL DNA Sequencer (Applied Biosystems).

PprA protein purification

Escherichia coli strain BL21(DE3) carrying pLysS was transformed with pET3pprAwt or pET3pprA446. In an effort to obtain a small-scale crude protein extract, IPTG-induced E. coli cells were harvested, resuspended in a buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM EDTA and 1 mM PMSF, and disrupted using the FastPROTEIN BLUE Kit (Qbiogene). The cell debris was removed by centrifugation, and the supernatant was used as the crude protein extract. For large-scale purification of the wild-type PprA protein, ITPG-induced E. coli cells were harvested, resuspended in buffer P1 containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA and 1 mM PMSF, and sonicated for 10 min on ice. After the cell debris was removed, polymin P was slowly added to the supernatant to give a final concentration of 0.5%. The suspension was stirred at 4°C for 30 min, after which time the precipitate was harvested by centrifugation, resuspended in buffer P2 containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA and 500 mM NaCl, and homogenized on ice. After centrifugation, ammonium sulphate was slowly added to the supernatant to 60% saturation. The suspension was stirred for 1 h and then centrifuged for 30 min at 4°C. The pellet was resuspended in buffer P3 containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA and 800 mM NaCl, and subsequently dialysed for 18 h against buffer P4 containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA and 300 mM NaCl. The protein was further purified to apparent homogeneity by passage through DEAE Sepharose CL-6B, HiPrep 16/60 Sephacryl S-300 and MonoQ HR5/5 columns (Amersham Biosciences). The purified fraction was concentrated and equilibrated with 20 mM Tris-HCl (pH 7.5).

Detection of intracellular levels of PprA and RecA

Deinococcus radiodurans cells were resuspended in 10 mM sodium phosphate buffer (pH 7.0) and irradiated with 2 kGy of γ-rays. Cells were then incubated in fresh TGY broth at 30°C for 2 h with agitation. The cells were harvested by centrifugation, resuspended in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1 mM PMSF and 1% SDS, and then disrupted using a FastPrep Cell Disruptor FP120 (Savant Instruments) with a FastPROTEIN BLUE Kit. The cell debris was removed by centrifugation, and the supernatant was subjected to SDS-PAGE analysis. The resolved proteins were transferred onto a PVDF membrane (Millipore), and incubated with E. coli RecA anti-serum (diluted 1:500) or D. radiodurans PprA anti-serum (1:10 000), together with alkaline phosphatase-conjugated rabbit IgG anti-serum (Applied Biosystems). As the sample loading control, E. coli GroEL anti-serum (1:4000) (StressGen Biotechnologies) was used to detect D. radiodurans GroEL. Chemiluminescent signals on the PVDF membrane were visualized using a Lumi-Imager F1 Workstation (Roche Diagnostics).

Acknowledgements

We thank Z. Alatas for transformation experiments and K. Yamamoto for critical discussions. This work was performed as part of Nuclear Energy Fundamentals Crossover Research, and financially supported by the Budget for Nuclear Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and based on screening and counselling by the Atomic Energy Commission of Japan.

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